Apo-2 ligand/trail formulations

ABSTRACT

The inventions include Apo2L/TRAIL formulations and methods of using such formulations. Lyophilized and crystal formulations of Apo-2L/TRAIL which are stable and have improved Apo2L/TRAIL trimer formation are provided. Methods of making Apo-2L/TRAIL formulations, as well as devices and kits containing such formulations are also provided.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 10/771,254filed Feb. 3, 2004 (now abandoned), which is a continuation-in-partapplication of international application PCT/US02/36251 (designating theUS) filed Nov. 12, 2002, which claims benefit of U.S. provisionalapplication No. 60/338,249 filed Nov. 13, 2001 (now abandoned), thecontents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates generally to Apo2L/TRAIL formulations. Inparticular, such Apo2L/TRAIL formulations include lyophilized andcrystal compositions.

BACKGROUND OF THE INVENTION

Various molecules, such as tumor necrosis factor-alpha (“TNF-alpha”),tumor necrosis factor-beta (“TNF-beta” or “lymphotoxin-alpha”),lymphotoxin-beta (“LT-beta”), CD30 ligand, CD27 CD40 ligand, OX-40ligand, 4-1BB ligand, Apo-1 ligand (also referred to as Fas ligand orCD95 ligand), Apo-2 ligand (also referred to as Apo2L or TRAIL), Apo-3ligand (also referred to as TWEAK), APRIL, OPG ligand (also referred toas RANK ligand, ODF, or TRANCE), and TALL-1 (also referred to as BlyS,BAFF or THANK) have been identified as members of the tumor necrosisfactor (“TNF”) family of cytokines [See, e.g., Gruss and Dower, Blood,85:3378-3404 (1995); Schmid et al., Proc. Natl. Acad. Sci., 83:1881(1986); Dealtry et al., Eur. J. Immunol., 17:689 (1987); Pitti et al.,J. Biol. Chem., 271:12687-12690 (1996); Wiley et al., Immunity,3:673-682 (1995); Browning et al., Cell, 72:847-856 (1993); Armitage etal. Nature, 357:80-82 (1992), WO 97/01633 published Jan. 16, 1997; WO97/25428 published Jul. 17, 1997; Marsters et al., Curr. Biol.,8:525-528 (1998); Chicheportiche et al., Biol. Chem., 272:32401-32410(1997); Hahne et al., J. Exp. Med., 188:1185-1190 (1998); WO98/28426published Jul. 2, 1998; WO98/46751 published Oct. 22, 1998; WO/98/18921published May 7, 1998; Moore et al., Science, 285:260-263 (1999); Shu etal., J. Leukocyte Biol., 65:680 (1999); Schneider et al., J. Exp. Med.,189:1747-1756 (1999); Mukhopadhyay et al., J. Biol. Chem.,274:15978-15981 (1999)]. Among these molecules, TNF-alpha, TNF-beta,CD30 ligand, 4-1BB ligand, Apo-1 ligand, Apo-2 ligand (Apo2L/TRAIL) andApo-3 ligand (TWEAK) have been reported to be involved in apoptotic celldeath.

Apo2L/TRAIL was identified several years ago as a member of the TNFfamily of cytokines. [see, e.g., Wiley et al., Immunity, 3:673-682(1995); Pitti et al., J. Biol. Chem., 271:12697-12690 (1996)] Thefull-length human Apo2L/TRAIL polypeptide is a 281 amino acid long, TypeII transmembrane protein. Some cells can produce a natural soluble formof the polypeptide, through enzymatic cleavage of the polypeptide'sextracellular region [Mariani et al., J. Cell. Biol., 137:221-229(1997)]. Crystallographic studies of soluble forms of Apo2L/TRAIL reveala homotrimeric structure similar to the structures of TNF and otherrelated proteins [Hymowitz et al., Molec. Cell, 4:563-571 (1999);Hymowitz et al., Biochemistry, 39:633-644 (2000)]. Apo2L/TRAIL, unlikeother TNF family members however, was found to have a unique structuralfeature in that three cysteine residues (at position 230 of each subunitin the homotrimer) together coordinate a zinc atom, and that the zincbinding is important for trimer stability and biological activity.[Hymowitz et al., supra; Bodmer et al., J. Biol. Chem., 275:20632-20637(2000)]

It has been reported in the literature that Apo2L/TRAIL may play a rolein immune system modulation, including autoimmune diseases such asrheumatoid arthritis, and in the treatment of HIV [see, e.g., Thomas etal., J. Immunol., 161:2195-2200 (1998); Johnsen et al., Cytokine,11:664-672 (1999); Griffith et al., J. Exp. Med., 189:1343-1353 (1999);Song et al., J. Exp. Med., 191:1095-1103 (2000); Jeremias et al., Eur.J. Immunol., 28:143-152 (1998); Katsikis et al., J. Exp. Med.,186:1365-1372 (1997); Miura et al., J. Exp. Med., 193:651-660 (2001)].

Soluble forms of Apo2L/TRAIL have also been reported to induce apoptosisin a variety of cancer cells in vitro, including colon, lung, breast,prostate, bladder, kidney, ovarian and brain tumors, as well asmelanoma, leukemia, and multiple myeloma [see, e.g., Wiley et al.,supra; Pitti et al., supra; Rieger et al., FEBS Letters, 427:124-128(1998); Ashkenazi et al., J. Clin. Invest., 104:155-162 (1999); Walczaket al., Nature Med., 5:157-163 (1999); Keane et al., Cancer Research,59:734-741 (1999); Mizutani et al., Clin. Cancer Res., 5:2605-2612(1999); Gazitt, Leukemia, 13:1817-1824 (1999); Yu et al., Cancer Res.,60:2384-2389 (2000); Chinnaiyan et al., Proc. Natl. Acad. Sci.,97:1754-1759 (2000)]. In vivo studies in murine tumor models furthersuggest that Apo2L/TRAIL, alone or in combination with chemotherapy orradiation therapy, can exert substantial anti-tumor effects [see, e.g.,Ashkenazi et al., supra; Walzcak et al., supra; Gliniak et al., CancerRes., 59:6153-6158 (1999); Chinnaiyan et al., supra; Roth et al.,Biochem. Biophys. Res. Comm., 265:1999 (1999)]. In contrast to manytypes of cancer cells, most normal human cell types appear to beresistant to apoptosis induction by certain recombinant forms ofApo2L/TRAIL [Ashkenazi et al., supra; Walzcak et al., supra]. Jo et al.has reported that a polyhistidine-tagged soluble form of Apo2L/TRAILinduced apoptosis in vitro in normal isolated human, but not non-human,hepatocytes [Jo et al., Nature Med., 6:564-567 (2000); see also, Nagata,Nature Med., 6:502-503 (2000)]. It is believed that certain recombinantApo2L/TRAIL preparations may vary in terms of biochemical properties andbiological activities on diseased versus normal cells, depending, forexample, on the presence or absence of a tag molecule, zinc content, and% trimer content [See, Lawrence et al., Nature Med., Letter to theEditor, 7:383-385 (2001); Qin et al., Nature Med., Letter to the Editor,7:385-386 (2001)].

Induction of various cellular responses mediated by such TNF familycytokines is believed to be initiated by their binding to specific cellreceptors. Previously, two distinct TNF receptors of approximately55-kDa (TNFR1) and 75-kDa (TNFR2) were identified [Hohman et al., J.Biol. Chem., 264:14927-14934 (1989); Brockhaus et al., Proc. Natl. Acad.Sci., 87:3127-3131 (1990); EP 417,563, published Mar. 20, 1991;Loetscher et al., Cell, 61:351 (1990); Schall et al., Cell, 61:361(1990); Smith et al., Science, 248:1019-1023 (1990); Lewis et al., Proc.Natl. Acad. Sci., 88:2830-2834 (1991); Goodwin et al., Mol. Cell. Biol.,11:3020-3026 (1991)]. Those TNFRs were found to share the typicalstructure of cell surface receptors including extracellular,transmembrane and intracellular regions. The extracellular portions ofboth receptors were found naturally also as soluble TNF-binding proteins[Nophar, Y. et al., EMBO J., 9:3269 (1990); and Kohno, T. et al., Proc.Natl. Acad. Sci. U.S.A., 87:8331 (1990); Hale et al., J. Cell. Biochem.Supplement 15F, 1991, p. 113 (P424)].

The extracellular portion of type 1 and type 2 TNFRs (TNFR1 and TNFR2)contains a repetitive amino acid sequence pattern of four cysteine-richdomains (CRDs) designated 1 through 4, starting from the NH₂-terminus.[Schall et al., supra; Loetscher et al., supra; Smith et al., supra;Nophar et al., supra; Kohno et al., supra; Banner et al., Cell,73:431-435 (1993)]. A similar repetitive pattern of CRDs exists inseveral other cell-surface proteins, including the p75 nerve growthfactor receptor (NGFR) [Johnson et al., Cell, 47:545 (1986); Radeke etal., Nature, 325:593 (1987)], the B cell antigen CD40 [Stamenkovic etal., EMBO J., 8:1403 (1989)], the T cell antigen OX40 [Mallet et al.,EMBO J., 9:1063 (1990)] and the Fas antigen [Yonehara et al., supra andItoh et al., Cell, 66:233-243 (1991)]. CRDs are also found in thesoluble TNFR (sTNFR)-like T2 proteins of the Shope and myxoma poxviruses[Upton et al., Virology, 160:20-29 (1987); Smith et al., Biochem.Biophys. Res. Commun., 176:335 (1991); Upton et al., Virology, 184:370(1991)]. Optimal alignment of these sequences indicates that thepositions of the cysteine residues are well conserved. These receptorsare sometimes collectively referred to as members of the TNF/NGFreceptor superfamily.

The TNF family ligands identified to date, with the exception oflymphotoxin-beta, are typically type II transmembrane proteins, whoseC-terminus is extracellular. In contrast, most receptors in the TNFreceptor (TNFR) family identified to date are typically type Itransmembrane proteins. In both the TNF ligand and receptor families,however, homology identified between family members has been foundmainly in the extracellular domain (“ECD”). Several of the TNF familycytokines, including TNF-alpha, Apo-1 ligand and CD40 ligand, arecleaved proteolytically at the cell surface; the resulting protein ineach case typically forms a homotrimeric molecule that functions as asoluble cytokine. TNF receptor family proteins are also usually cleavedproteolytically to release soluble receptor ECDs that can function asinhibitors of the cognate cytokines.

Pan et al. have disclosed another TNF receptor family member referred toas “DR4” [Pan et al., Science, 276:111-113 (1997); see also WO98/32856published Jul. 30, 1998]. The DR4 was reported to contain a cytoplasmicdeath domain capable of engaging the cell suicide apparatus. Pan et al.disclose that DR4 is believed to be a receptor for the ligand known asApo2L/TRAIL.

In Sheridan et al., Science, 277:818-821 (1997) and Pan et al., Science,277:815-818 (1997), another molecule believed to be a receptor forApo2L/TRAIL is described [see also, WO98/51793 published Nov. 19, 1998;WO98/41629 published Sep. 24, 1998]. That molecule is referred to as DR5(it has also been alternatively referred to as Apo-2; TRAIL-R, TR6,Tango-63, hAP08, TRICK2 or KILLER [Screaton et al., Curr. Biol.,7:693-696 (1997); Walczak et al., EMBO J., 16:5386-5387 (1997); Wu etal., Nature Genetics, 17:141-143 (1997); WO98/35986 published Aug. 20,1998; EP870,827 published Oct. 14, 1998; WO98/46643 published Oct. 22,1998; WO99/02653 published Jan. 21, 1999; WO99/09165 published Feb. 25,1999; WO99/11791 published Mar. 11, 1999]. Like DR4, DR5 is reported tocontain a cytoplasmic death domain and be capable of signalingapoptosis. The crystal structure of the complex formed betweenApo-2L/TRAIL and DR5 is described in Hymowitz et al., Molecular Cell,4:563-571 (1999).

A further group of recently identified receptors are referred to as“decoy receptors,” which are believed to function as inhibitors, ratherthan transducers of signaling. This group includes DCR1 (also referredto as TRID, LIT or TRAIL-R3) [Pan et al., Science, 276:111-113 (1997);Sheridan et al., Science, 277:818-821 (1997); McFarlane et al., J. Biol.Chem., 272:25417-25420 (1997); Schneider et al., FEBS Letters,416:329-334 (1997); Degli-Esposti et al., J. Exp. Med., 186:1165-1170(1997); and Mongkolsapaya et al., J. Immunol., 160:3-6 (1998)] and DCR2(also called TRUNDD or TRAIL-R4) [Marsters et al., Curr. Biol.,7:1003-1006 (1997); Pan et al., FEBS Letters, 424:41-45 (1998);Degli-Esposti et al., Immunity, 7:813-820 (1997)], both cell surfacemolecules, as well as OPG [Simonet et al., supra; Emery et al., infra]and DCR3 [Pitti et al., Nature, 396:699-703 (1998)], both of which aresecreted, soluble proteins. Apo2L/TRAIL has been reported to bind thosereceptors referred to as DcR1, DcR2 and OPG.

Apo2L/TRAIL is believed to act through the cell surface “deathreceptors” DR4 and DR5 to activate caspases, or enzymes that carry outthe cell death program. Upon ligand binding, both DR4 and DR5 cantrigger apoptosis independently by recruiting and activating theapoptosis initiator, caspase-8, through the death-domain-containingadaptor molecule referred to as FADD/Mort1 [Kischkel et al., Immunity,12:611-620 (2000); Sprick et al., Immunity, 12:599-609 (2000); Bodmer etal., Nature Cell Biol., 2:241-243 (2000)]. In contrast to DR4 and DR5,the DcR1 and DcR2 receptors do not signal apoptosis.

For a review of the TNF family of cytokines and their receptors, seeAshkenazi and Dixit, Science, 281:1305-1308 (1998); Ashkenazi and Dixit,Curr. Opin. Cell Biol., 11:255-260 (2000); Golstein, Curr. Biol.,7:750-753 (1997); Gruss and Dower, supra; Nagata, Cell, 88:355-365(1997); Locksley et al., Cell, 104:487-501 (2001).

SUMMARY OF THE INVENTION

Certain proteins, such as Apo2L/TRAIL and other members of the TNFfamily of cytokines, exhibit biological activity when the protein is ina trimer or trimeric form. Thus, for purposes of therapeutic or evendiagnostic use, formulations of such proteins are desired wherein theprotein is stable and remains biologically active, particularly stablein a trimeric form. Applicants have found that certain formulationcomponents, or “excipients”, can provide stability for such proteinslike Apo2L/TRAIL and enhance solubility (i.e., to reduce aggregation orprecipitation of the protein). Applicants also surprisingly found that,under certain conditions, Apo2L/TRAIL can readily crystallize. Suchcrystal forms of Apo2L/TRAIL may be useful in preparation of suspensionformulations of Apo2L/TRAIL and/or provide an effective and efficientprocess for protein purification.

Accordingly, the present invention provides compositions or formulationscomprising Apo2L/TRAIL and one or more excipients which providesufficient ionic strength to enhance solubility and/or stability of theApo2L/TRAIL, wherein the composition optionally has a pH of 6 (or about6) to 9 (or about 9). Optionally, the excipient(s) providing sufficientionic strength is salt, and may comprise an arginine salt or sulfatesalt. In one embodiment, the compositions may further comprise a buffer.Optionally, the concentration of the Apo2L/TRAIL protein in thecomposition is about 1 to about 100 mg/ml, about 1 to about 20 mg/ml,about 10 to about 20 mg/ml, or about 20 mg/ml. The compositions of theinvention may comprise liquid formulations or lyophilized formulations.The compositions may also comprise suspension formulations in which theApo2L/TRAIL protein is in the form of crystals. Optionally, it may bedesirable to include one or more surfactants or other excipients in thecomposition. Such surfactants may, for instance, comprise a polysorbateor poloxamer. Particularly desired formulations are those in which theexcipient(s) provide for optimized Apo2L/TRAIL trimer content andminimize the amount of Apo2L/TRAIL dimer or aggregate formation.Optionally, the formulations contain no more than 10% Apo2L/TRAIL dimeror 5% Apo2L/TRAIL aggregates (of the total amount of Apo2L/TRAIL proteinin the formulation).

In optional embodiments, the present invention provides compositionscomprising about 1 to about 20 mg/ml of Apo2L/TRAIL and arginine salt,wherein the composition has a pH of about 6.5 to about 8.5. Optionally,the compositions further comprise a buffer such as Tris and a surfactantsuch as polysorbate. Optionally, the Apo2L/TRAIL protein does notinclude (i.e., is not linked or fused to) any epitope tag molecule(s) orleucine zipper molecule(s).

The present invention provides compositions comprising about 1 to about20 mg/ml of Apo2L/TRAIL, about 0.4 to about 0.5M arginine salt, andbuffer, wherein the compositions have a pH of about 7 to about 7.5. TheApo2L/TRAIL protein may be human Apo2L/TRAIL protein comprising aminoacid residues 114 to 281 of FIG. 1. Optionally, the Apo2L/TRAIL proteinis recombinantly expressed in host cells such as E. coli.

In addition, the invention provides methods for preparing thecompositions described above. In the methods, the compositions areprepared by admixing or combining Apo2L/TRAIL and one or more excipientswhich provide sufficient ionic strength to enhance solubility and/orstability of the Apo2L/TRAIL, wherein the composition has a pH of 6 (orabout 6) to 9 (or about 9). Optionally, the excipient(s) providingsufficient ionic strength is salt, and may comprise an arginine salt orsulfate salt. A buffer may also be included to maintain the pH of thecomposition, and optionally to maintain the pH at about 6.5 to about7.5. Optionally, the concentration of the Apo2L/TRAIL protein in theformulation is about 1 to about 100 mg/ml, about 1 to about 20 mg/ml,about 10 to about 20 mg/ml, or at least 20 mg/ml. In particularlydesirable embodiments, the resulting compositions are pharmaceuticallyacceptable formulations.

In further embodiments, the invention provides compositions comprisingApo2L/TRAIL protein crystals. Preferably, the Apo2L/TRAIL proteincrystal compositions contain 5% (or about 5%) to 10% (or about 10%)water. Optionally, the Apo2L/TRAIL protein crystal compositions maycontain one or more excipients such as sucrose, trehalose, arginine,Tween 20, or Captisol™.

In still further embodiments, the invention provides methods of makingcompositions comprising Apo2L/TRAIL crystals.

In yet further embodiments, the invention provides methods of making andpurifying Apo2L/TRAIL.

In additional embodiments, the invention provides kits comprising:

-   -   (a) a container comprising an Apo2L/TRAIL composition described        herein and    -   (b) instructions for using the Apo2L/TRAIL composition; such as        for using the composition to treat a disorder against which the        composition is effective. Optionally, the disorder is cancer,        and more particularly, is a breast, lung, or colon (or        colorectal) cancer.

In still further aspects, the invention provides methods for treating adisorder, such as cancer or an immune related disorder, in a mammalcomprising administering to the mammal, optionally by either injectionor infusion, an effective amount of an Apo2L/TRAIL composition providedby the present invention.

In more particular embodiments of the invention the following areprovided:

A stable formulation of Apo-2 ligand, comprising Apo-2 ligand and about0.2M to about 0.5M salt, wherein said formulation has a pH of about 6 toabout 9. Optionally, the salt is an arginine salt or sodium sulphate.Optionally, the concentration of the arginine salt in the formulation isabout 0.4M to about 0.5 M. The arginine salt may include argininesuccinate, arginine sulphate, arginine malate, arginine citrate,arginine tartrate, or arginine phosphate. The Apo-2 ligand mayoptionally comprise crystallized protein. The formulation may comprise alyophilized or suspension formulation. Optionally, the pH of theformulation is about 6.5 to about 8.5, and optionally, about 7 to about7.5. Optionally, the formulation further comprises surfactant, such as apolysorbate or poloxamer. Optionally, the concentration of thesurfactant in the formulation is about 0.005% to about 0.2%. Optionally,the formulation further comprises buffer, such as Tris buffer or Hepes.Optionally, the formulation further comprises one or more divalent metalions or a preservative. Optionally, the formulation is storage-stablefor at least 12 months.

A stable, lyophilized formulation of Apo-2 ligand, comprising about 1mg/ml to about 20 mg/ml Apo-2 ligand, about 0.2 M to about 0.5M argininesalt, buffer, and surfactant, wherein said formulation has a pH of about6 to about 9. Optionally, the arginine salt is arginine succinate, andthe concentration of the arginine succinate may be about 0.4M to about0.5M. Optionally, the buffer is Tris buffer, and the surfactant is apolysorbate. Optionally, the formulation further comprises one or moredivalent metal ions.

A stable formulation of Apo-2 ligand, comprising about 1 mg/ml to about20 mg/ml Apo-2 ligand, about 0.2M to about 0.5 M salt, buffer, andsurfactant, wherein said Apo-2 ligand comprises crystallized protein andsaid formulation has a pH of about 6 to about 9. Optionally, the salt issodium sulphate, and the buffer is Tris buffer. Optionally, thesurfactant is polysorbate, and the pH is about 7 to about 7.5.

A stable formulation of Apo-2 ligand, comprising about 0.1 mg/ml toabout 2 mg/ml Apo-2 ligand, sugar, and surfactant, wherein saidformulation has a pH of about 6 to about 9. Optionally, the sugar istrehalose, and the concentration of the sugar in the formulation may beabout 1% to about 8%. Optionally, the formulation is lyophilized.

A stable Apo2L/TRAIL protein crystal composition, comprising about 5% toabout 10% water. Optionally, the Apo2L/TRAIL protein crystal compositioncomprises one or more excipients such as sucrose, trehalose, arginine,Tween 20, or Captisol™.

A method of making a stable formulation of Apo-2 ligand, comprisingsteps of (a) providing about 1 mg/ml to about 20 mg/ml Apo-2 ligand,about 0.2 M to about 0.5M arginine salt, buffer, and surfactant, (b)combining or mixing the ingredients of step (a) to make a formulation,and (c) adjusting the pH of the formulation of step (b) to about 6 toabout 9. Optionally, the arginine salt is arginine succinate, and theconcentration of the arginine succinate is about 0.4M to about 0.5M.Optionally, the buffer is Tris buffer, and the surfactant is apolysorbate.

A method of making crystallized Apo-2 ligand, comprising steps of (a)providing Apo-2 ligand, buffer, and monovalent cationic salt, (b)combining or mixing the ingredients of step (a) to make a formulation ata temperature of about 20° C. to about 30° C., and (c) lowering thetemperature of the formulation of step (b) to about 2° C. to about 8°C.; wherein Apo-2 ligand crystallization occurs as the temperature ofthe formulation of step (b) is lowered. Optionally, the salt is sodiumsulphate or sodium chloride. The concentration of the salt may be about0.1M to about 0.15M. Optionally, the formulation of step (b) is agitatedas the temperature is lowered in step (c). Optionally, the methodfurther comprises a step (d) in which the Apo-2 ligand crystals aredried. Optionally, prior to the step (d), the Apo-2 ligand crystals arewashed.

A method of making Apo-2 ligand, comprising the steps of: (a) providinghost cells comprising a vector containing DNA encoding Apo-2 ligand; (b)culturing the host cells in culture medium under conditions sufficientto express Apo-2 ligand; (c) obtaining said expressed Apo-2 ligand fromthe host cells and culture medium; (d) formulating said Apo-2 ligandinto a solution containing sodium chloride or sodium sulphate to make aformulation at a temperature of about 20° C. to about 30° C., and (e)lowering the temperature of said formulation of step (d) to about 2° C.to about 8° C., wherein Apo-2 ligand crystals form when the temperatureof step (e) is lowered. Optionally, prior to step (d), the Apo-2 ligandprotein is concentrated, and the protein may be concentrated bycentrifugation, column chromatography or ultrafiltration. Optionally,step (d) is conducted by applying the Apo-2 ligand to a chromatographiccolumn (such as a cation exchange column) and eluting the Apo-2 ligandinto a sodium chloride or sodium sulphate containing buffer. Optionally,step (d) is further conducted by including polyethelene glycol to thepool as the chromatography is performed. Optionally, the cation exchangecolumn comprises SP-Sepharose fast flow, CM-Sepharose fast flow, orMacro-prep ceramic HS, and the buffer solution contains 50 mM Hepes, 50mM Tris, 50 mM triethanolamine, 0.05% Triton X 100, 1 mM DTT, pH7.5-8.0. Optionally, the formulation is agitated during step (e).Optionally, the pH of the formulation in step (d) is about 6.5 to about8.5. Optionally, the host cells are prokaryote cells, such as E. coli.

A device for administering a formulation of Apo-2 ligand to a mammal,comprising a container holding at least one dosage unit of the Apo-2ligand formulations described herein. Optionally, the device is a peninjector device, and the container is a cartridge.

An article of manufacture, comprising a container which includes anApo2L/TRAIL formulation described herein, and printed instructions foruse of the Apo-2L/TRAIL formulation. Optionally, the container is abottle, vial, syringe, or test tube. Optionally, the article ofmanufacture comprises a second container which includeswater-for-injection, saline, Ringer's solution, or dextrose solution.

A method of inducing apoptosis in mammalian cells, comprising exposingmammalian cells to an effective amount of an Apo-2 ligand formulationdescribed herein. The mammalian cells may be cancer cells.

A method of treating cancer in a mammal, comprising administering to amammal diagnosed as having cancer an effective amount of an Apo-2 ligandformulation described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of human Apo-2L/TRAIL cDNA (SEQ IDNO:2) and its derived amino acid sequence (SEQ ID NO:1). The “N” atnucleotide position 447 (in SEQ ID NO:2) is used to indicate thenucleotide base may be a “T” or “G”.

FIG. 2 shows data (% trimer remaining and % IEX main peak remaining) forvarious Apo2L/TRAIL formulations after 1 week storage at 30° C.

FIG. 3A shows data (% trimer remaining and % IEX main peak remaining)for various Apo2L/TRAIL formulations after 4 months storage at 40° C.

FIG. 3B shows data (% trimer remaining, % monomer, and % IEX main peakremaining) for various Apo2L/TRAIL formulations after 1 month storage at50° C.

FIG. 3C shows an Arrhenius plot predictive of shelf-life for thedescribed Apo2L/TRAIL formulation.

FIGS. 4A-4B show graphs of % bioactivity and % trimer of two differentformulations at varying pH.

FIG. 5 illustrates the structure of Apo2L/TRAIL and coordination of thestructure by an intrinsic zinc molecule.

FIG. 6 shows the effects of varying concentrations of polysorbate onstability of an Apo2L/TRAIL formulation.

FIG. 7 shows the effects of varying concentrations of zinc on stabilityof an Apo2L/TRAIL formulation.

FIG. 8 shows the equilibrium solubility and crystallization ofApo2L/TRAIL in a sodium sulphate formulation.

FIG. 9 shows the equilibrium solubility and crystallization ofApo2L/TRAIL in various salt concentrations.

FIG. 10A shows the effects of agitation rates on crystallization ofApo2L/TRAIL.

FIG. 10B shows the dissolution profile of Apo2L/TRAIL crystals underagitation.

FIG. 10C shows the effects of agitation rate on Apo2L/TRAIL crystal sizedistribution.

FIG. 11A shows the IEX profile of the Apo2L/TRAIL after reconstitutionof vacuum dried crystals.

FIG. 11B shows the bioactivity of the Apo2L/TRAIL after reconstitutionof the vacuum dried crystals.

FIG. 12 shows an Arrhenius plot predictive of shelf-life for thedescribed Apo2L/TRAIL formulation.

FIG. 13 shows a SDS-PAGE silver stain gel illustrating purity of thedescribed Apo2L/TRAIL preparations.

FIG. 14 shows the effects of various salts on crystallization ofApo2L/TRAIL.

FIGS. 15A-15C show the effects of various excipients on the stability oflyophilized Apo2L/TRAIL crystals.

FIGS. 16A-16B show the effects of residual moisture content on thestability of lyophilized Apo2L/TRAIL crystals.

FIGS. 17A-17B show the relative amounts of reducible and non-reducibledimers formed upon storage of lyophilized Apo2L/TRAIL crystalscontaining 2.5% and 12% water.

FIGS. 18A-18B show the relative amounts of covalent dimer species, bothreducible and non-reducible, contained in the apparent larger molecularweight species formed upon storage of lyophilized Apo2L/TRAIL crystalscontaining 2.5% and 12% water.

FIGS. 19A-19B show the relationship between the residual moisturecontent of the crystals and the pseudo-first order rate constants ofcovalent hexamer and disulfide-linked dimer formation at 50° C. and 40°C.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

“TNF family member” is used in a broad sense to refer to variouspolypeptides that share some similarity to tumor necrosis factor (TNF)with respect to structure or function. Certain structural and functionalcharacteristics associated with the TNF family of polypeptides are knownin the art and described, for example, in the above Background of theInvention. Such polypeptides include but are not limited to thosepolypeptides referred to in the art as TNF-alpha, TNF-beta, CD40 ligand,CD30 ligand, CD27 ligand, OX-40 ligand, 4-1BB ligand, Apo-1 ligand (alsoreferred to as Fas ligand or CD95 ligand), Apo-2L/TRAIL (also referredto as TRAIL), Apo-3 ligand (also referred to as TWEAK), APRIL, OPGligand (also referred to as RANK ligand, ODF, or TRANCE), and TALL-1(also referred to as BlyS, BAFF or THANK) (See, e.g., Gruss and Dower,Blood 1995, 85:3378-3404; Pitti et al., J. Biol. Chem. 1996,271:12687-12690; Wiley et al., Immunity 1995, 3:673-682; Browning etal., Cell 1993, 72:847-856; Armitage et al. Nature 1992, 357:80-82, PCTPublication Nos. WO 97/01633; and WO 97/25428; Marsters et al., Curr.Biol. 1998, 8:525-528; Chicheportiche et al., Biol. Chem. 1997,272:32401-32410; Hahne et al., J. Exp. Med. 1998, 188:1185-1190; PCTPublication Nos. WO98/28426; WO98/46751; and WO/98/18921; Moore et al.,Science 1999, 285:260-263; Shu et al., J. Leukocyte Biol. 1999, 65:680;Schneider et al., J. Exp. Med. 1999, 189:1747-1756; Mukhopadhyay et al.,J. Biol. Chem. 1999, 274:15978-15981).

The terms “Apo2L/TRAIL”, “Apo2L”, “Apo-2 ligand” and “TRAIL” are usedherein to refer to a polypeptide sequence which includes amino acidresidues 114-281, inclusive, 95-281, inclusive, residues 92-281,inclusive, residues 91-281, inclusive, residues 41-281, inclusive,residues 15-281, inclusive, or residues 1-281, inclusive, of the aminoacid sequence shown in FIG. 1 (SEQ ID NO:1), as well as biologicallyactive fragments, deletional, insertional, or substitutional variants ofthe above sequences. In one embodiment, the polypeptide sequencecomprises residues 114-281 of FIG. 1 (SEQ ID NO:1), and optionally,consists of residues 114-281 of FIG. 1 (SEQ ID NO:1). Optionally, thepolypeptide sequence comprises residues 92-281 or residues 91-281 ofFIG. 1 (SEQ ID NO:1). The Apo-2L polypeptides may be encoded by thenative nucleotide sequence shown in FIG. 1 (SEQ ID NO:2). Optionally,the codon which encodes residue Proll9 (FIG. 1; SEQ ID NO:2) may be“CCT” or “CCG”. In other embodiments, the fragments or variants arebiologically active and have at least about 80% amino acid sequenceidentity, more preferably at least about 90% sequence identity, and evenmore preferably, at least 95%, 96%, 97%, 98%, or 99% sequence identitywith any one of the above recited Apo2L/TRAIL sequences. Optionally, theApo2L/TRAIL polypeptide is encoded by a nucleotide sequence whichhybridizes under stringent conditions with the encoding polynucleotidesequence provided in FIG. 1 (SEQ ID NO:2). The definition encompassessubstitutional variants of Apo2L/TRAIL in which at least one of itsnative amino acids are substituted by an alanine residue. Particularsubstitutional variants of the Apo2L/TRAIL include those in which atleast one amino acid is substituted by an alanine residue. Thesesubstitutional variants include those identified, for example, as“D203A”; “D218A” and “D269A.” This nomenclature is used to identifyApo2L/TRAIL variants wherein the aspartic acid residues at positions203, 218, and/or 269 (using the numbering shown in FIG. 1 (SEQ ID NO:1))are substituted by alanine residues. Optionally, the Apo2L variants maycomprise one or more of the alanine substitutions which are recited inTable I of published PCT application WO 01/00832. Substitutionalvariants include one or more of the residue substitutions identified inTable I of WO 01/00832 published Jan. 4, 2001. The definition alsoencompasses a native sequence Apo2L/TRAIL isolated from an Apo2L/TRAILsource or prepared by recombinant or synthetic methods. The Apo2L/TRAILof the invention includes the polypeptides referred to as Apo2L/TRAIL orTRAIL disclosed in PCT Publication Nos. WO97/01633 and WO97/25428. Theterms “Apo2L/TRAIL” or “Apo2L” are used to refer generally to forms ofthe Apo2L/TRAIL which include monomer, dimer or trimer forms of thepolypeptide. All numbering of amino acid residues referred to in theApo2L sequence use the numbering according to FIG. 1 (SEQ ID NO:1),unless specifically stated otherwise. For instance, “D203” or “Asp203”refers to the aspartic acid residue at position 203 in the sequenceprovided in FIG. 1 (SEQ ID NO:1).

The term “Apo2L/TRAIL extracellular domain” or “Apo2L/TRAIL ECD” refersto a form of Apo2L/TRAIL which is essentially free of transmembrane andcytoplasmic domains. Ordinarily, the ECD will have less than 1% of suchtransmembrane and cytoplasmic domains, and preferably, will have lessthan 0.5% of such domains. It will be understood that any transmembranedomain(s) identified for the polypeptides of the present invention areidentified pursuant to criteria routinely employed in the art foridentifying that type of hydrophobic domain. The exact boundaries of atransmembrane domain may vary but most likely by no more than about 5amino acids at either end of the domain as initially identified. Inpreferred embodiments, the ECD will consist of a soluble, extracellulardomain sequence of the polypeptide which is free of the transmembraneand cytoplasmic or intracellular domains (and is not membrane bound).Particular extracellular domain sequences of Apo-2L/TRAIL are describedin PCT Publication Nos. WO97/01633 and WO97/25428.

The term “Apo2L/TRAIL monomer” or “Apo2L monomer” refers to a covalentchain of an extracellular domain sequence of Apo2L.

The term “Apo2L/TRAIL dimer” or “Apo2L dimer” refers to two Apo-2Lmonomers joined in a covalent linkage via a disulfide bond. The term asused herein includes free standing Apo2L dimers and Apo2L dimers thatare within trimeric forms of Apo2L (i.e., associated with another, thirdApo2L monomer).

The term “Apo2L/TRAIL trimer” or “Apo2L trimer” refers to three Apo2Lmonomers that are non-covalently associated.

The term “Apo2L/TRAIL aggregate” is used to refer to self-associatedhigher oligomeric forms of Apo2L/TRAIL, such as Apo2L/TRAIL trimers,which form, for instance, hexameric and nanomeric forms of Apo2L/TRAIL.

Determination of the presence and quantity of Apo2L/TRAIL monomer,dimer, or trimer (or other aggregates) may be made using methods andassays known in the art (and using commercially available materials),such as native size exclusion HPLC (“SEC”), denaturing size exclusionusing sodium dodecyl sulphate (“SDS-SEC”), reverse phase HPLC, capillaryelectrophoresis, and including those methods described in further detailin the Examples below.

The term “tagged” when used herein refers to a chimeric polypeptidecomprising Apo2L/TRAIL, or a portion thereof, fused to a “tagpolypeptide”. The tag polypeptide has enough residues to provide anepitope against which an antibody can be made or to provide some otherfunction, such as metal ion chelation, yet is short enough such that itgenerally does not interfere with activity of the TNF family cytokine.The tag polypeptide preferably also is fairly unique so that atag-specific antibody does not substantially cross-react with otherepitopes. Suitable tag polypeptides generally have at least six aminoacid residues and usually between about 8 to about 50 amino acidresidues (preferably, between about 10 to about 20 residues).

The term “divalent metal ion” refers to a metal ion having two positivecharges. Examples of divalent metal ions include but are not limited tozinc, cobalt, nickel, cadmium, magnesium, and manganese. Particularforms of such metals that may be employed include salt forms (e.g.,pharmaceutically acceptable salt forms), such as chloride, acetate,carbonate, citrate and sulfate forms of the above mentioned divalentmetal ions. Optionally, a divalent metal ion for use in the presentinvention is zinc, and preferably, the salt form, zinc sulfate or zincchloride.

“Isolated,” when used to describe the various proteins disclosed herein,means protein that has been identified and separated and/or recoveredfrom a component of its natural environment. Contaminant components ofits natural environment are materials that would typically interferewith diagnostic or therapeutic uses for the protein, and may includeenzymes, hormones, and other proteinaceous or non-proteinaceous solutes.In preferred embodiments, the protein will be purified (1) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator, or (2) tohomogeneity by SDS-PAGE under non-reducing or reducing conditions usingCoomassie blue or, preferably, silver stain, or (3) to homogeneity bymass spectroscopic or peptide mapping techniques. Isolated proteinincludes protein in situ within recombinant cells, since at least onecomponent of the Apo2L/TRAIL natural environment will not be present.Ordinarily, however, isolated protein will be prepared by at least onepurification step.

An “isolated” Apo2L/TRAIL nucleic acid molecule is a nucleic acidmolecule that is identified and separated from at least one contaminantnucleic acid molecule with which it is ordinarily associated in thenatural source of the Apo2L/TRAIL nucleic acid. An isolated Apo2L/TRAILnucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated Apo2L/TRAIL nucleic acid moleculestherefore are distinguished from the Apo2L/TRAIL nucleic acid moleculeas it exists in natural cells. However, an isolated Apo2L/TRAIL nucleicacid molecule includes Apo2L/TRAIL nucleic acid molecules contained incells that ordinarily express Apo2L/TRAIL where, for example, thenucleic acid molecule is in a chromosomal location different from thatof natural cells.

“Percent (%) amino acid sequence identity” with respect to the sequencesidentified herein is defined as the percentage of amino acid residues ina candidate sequence that are identical with the amino acid residues inthe Apo2L/TRAIL sequence, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity,and not considering any conservative substitutions as part of thesequence identity. Alignment for purposes of determining percent aminoacid sequence identity can be achieved in various ways that are withinthe skill in the art can determine appropriate parameters for measuringalignment, including assigning algorithms needed to achieve maximalalignment over the full-length sequences being compared. For purposesherein, percent amino acid identity values can be obtained using thesequence comparison computer program, ALIGN-2, which was authored byGenentech, Inc. and the source code of which has been filed with userdocumentation in the US Copyright Office, Washington, D.C., 20559,registered under the US Copyright Registration No. TXU510087. TheALIGN-2 program is publicly available through Genentech, Inc., South SanFrancisco, Calif. All sequence comparison parameters are set by theALIGN-2 program and do not vary.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA tore-anneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired identitybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“High stringency conditions”, as defined herein, are identified by thosethat: (1) employ low ionic strength and high temperature for washing;0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecylsulfate at 50° C.; (2) employ during hybridization a denaturing agent;50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include overnight incubation at 37° C. ina solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmonsperm DNA, followed by washing the filters in 1×SSC at about 37-50° C.The skilled artisan will recognize how to adjust the temperature, ionicstrength, etc. as necessary to accommodate factors such as probe lengthand the like.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “storage-stable” is used to describe a formulation having ashelf-life acceptable for a product in the distribution chain ofcommerce, for instance, at least 12 months at a given temperature, andpreferably, at least 24 months at a given temperature. Optionally, sucha storage-stable formulation contains no more than 5% aggregates, nomore than 10% dimers, and/or minimal changes in charge heterogeneity orbiological activity. Degradation pathways for proteins can involvechemical instability (i.e. any process which involves modification ofthe protein by bond formation or cleavage resulting in a new chemicalentity) or physical instability (i.e. changes in the higher orderstructure of the protein). Chemical instability can result from, forexample, deamidation, racemization, hydrolysis, oxidation, betaelimination or disulfide exchange. Physical instability can result from,for example, denaturation, aggregation, precipitation or adsorption. Thethree most common protein degradation pathways are protein aggregation,deamidation and oxidation. Cleland et al. Critical Reviews inTherapeutic Drug Carrier Systems 10(4): 307-377 (1993).

As used herein, “soluble” refers to polypeptides that, when in aqueoussolutions, are completely dissolved, resulting in a clear to slightlyopalescent solution with no visible particulates, as assessed by visualinspection. A further assay of the turbidity of the solution (orsolubility of the protein) may be made by measuring UV absorbances at340 nm to 360 nm with a 1 cm pathlength cell where turbidity at 20 mg/mlis less than 0.05 absorbance units.

An “osmolyte” refers to a tonicity modifier or osmotic adjuster thatlends osmolality to a solution. Osmolality refers to the total osmoticactivity contributed by ions and nonionized molecules to a solution.Examples include inorganic salts such as sodium chloride, polyethyleneglycols (PEGs), polypropylene glycol, sugars such as sucrose ortrehalose, glycerol, amino acids, and sugar alcohols such as mannitolknown to the art that are generally regarded as safe (GRAS).

“Preservatives” can act to prevent bacteria, viruses, and fungi fromproliferating in the formulation, and anti-oxidants, or other compoundscan function in various ways to preserve the stability of theformulation. Examples include octadecyldimethylbenzyl ammonium chloride,hexamethonium chloride, benzalkonium chloride (a mixture ofalkylbenzyldimethylammonium chlorides in which the alkyl groups arelong-chain compounds), and benzethonium chloride. Other types ofcompounds include aromatic alcohols such as phenol and benzyl alcohol,alkyl parabens such as methyl or propyl paraben, and m-cresol.Optionally, such compound is phenol or benzyl alcohol. The preservativeor other compound will optionally be included in a liquid or aqueousform of the Apo2L/TRAIL formulation, but not usually in a lyophilizedform of the formulation. In the latter case, the preservative or othercompound will typically be present in the water for injection (WFI) orbacteriostatic water for injection (BWFI) used for reconstitution.

A “surfactant” can act to decrease turbidity or denaturation of aprotein in a formulation. Examples of surfactants include non-ionicsurfactant such as a polysorbate, e.g., polysorbates 20, 60, or 80, apoloxamer, e.g., poloxamer 184 or 188, Pluronic polyols,ethylene/propylene block polymers or any others known to the art thatare GRAS. Optionally, the surfactant is a polysorbate or poloxamer.

A “buffer” as used herein is any suitable buffer that is GRAS andgenerally confers a pH from about 6 to about 9, optionally from about6.5 to about 8.5, and optionally at about 7 to about 7.5, if thepolypeptide is Apo2L/TRAIL. Examples include Tris, Hepes,triethanolamine, histidine, or any others known to the art to have thedesired effect.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors; platelet-growth factor;transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-likegrowth factor-I and -II; erythropoietin (EPO); osteoinductive factors;interferons such as interferon-α, -β, and -gamma; colony stimulatingfactors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-11, IL-12; and other polypeptide factors including LIFand kit ligand (KL). As used herein, the term cytokine includes proteinsfrom natural sources or from recombinant cell culture and biologicallyactive equivalents of the native sequence cytokines.

The term, “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g., I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,ranimustine; antibiotics such as the enediyne antibiotics (e.g.calicheamicin, especially calicheamicin gamma1I and calicheamicin phiI1,see, Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994); dynemicin,including dynemicin A; bisphosphonates, such as clodronate; anesperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromomophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(Adriamycin™) (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol;nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (TAXOTER®, Rhone-Poulenc Rorer, Antony, France); chlorambucil;gemcitabine (Gemzar™); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;vinorelbine (Navelbine™); novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomeraseinhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such asretinoic acid; capecitabine; and pharmaceutically acceptable salts,acids or derivatives of any of the above. Also included in thisdefinition are anti-hormonal agents that act to regulate or inhibithormone action on tumors such as anti-estrogens and selective estrogenreceptor modulators (SERMs), including, for example, tamoxifen(including Nolvadex™), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene(Fareston™); aromatase inhibitors that inhibit the enzyme aromatase,which regulates estrogen production in the adrenal glands, such as, forexample, 4(5)-imidazoles, aminoglutethimide, megestrol acetate(Megace™), exemestane, formestane, fadrozole, vorozole (Rivisor™),letrozole (Femara™) and anastrozole (Arimidex™); and anti-androgens suchas flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially cancer celloverexpressing any of the genes identified herein, either in vitro or invivo. Thus, the growth inhibitory agent is one which significantlyreduces the percentage of cells overexpressing such genes in S phase.Examples of growth inhibitory agents include agents that block cellcycle progression (at a place other than S phase), such as agents thatinduce G1 arrest and M-phase arrest. Classical M-phase blockers includethe vincas (vincristine and vinblastine), taxol, and topo II inhibitorssuch as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.Those agents that arrest G1 also spill over into S-phase arrest, forexample, DNA alkylating agents such as tamoxifen, prednisone,dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil,and ara-C. Further information can be found in The Molecular Basis ofCancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycleregulation, oncogens, and antineoplastic drugs” by Murakami et al., (WBSaunders: Philadelphia, 1995), especially p. 13.

“Biologically active” or “biological activity” for the purposes hereinmeans (a) having the ability to induce or stimulate apoptosis in atleast one type of mammalian cancer cell or virally-infected cell in vivoor ex vivo, either alone as a single agent or in combination with achemotherapeutic agent (b) capable of raising an antibody, i.e.,immunogenic; (c) capable of binding and/or stimulating a receptor forApo2L/TRAIL (such receptors may include the DR4 receptor, DR5 receptor,OPG, DcR1 receptor, and DcR2 receptor); or (d) retaining the activity ofa native or naturally-occurring Apo2L/TRAIL polypeptide. Assays fordetermining biological activity of the Apo2L/TRAIL can be conductedusing methods known in the art, such as DNA fragmentation (see, e.g.,Marsters et al., Curr. Biology, 6: 1669 (1996)), caspase inactivation,DR4 binding, DR5 binding (see, e.g., WO 98/51793, published Nov. 19,1998), DcR1 binding (see, e.g., WO 98/58062, published Dec. 23, 1998),DcR2 binding (see, e.g., WO 99/10484, published Mar. 4, 1999) as well asthe assays described in PCT Publication Nos. WO97/01633, WO97/25428, WO01/00832, and WO 01/22987.

The terms “apoptosis” and “apoptotic activity” are used in a broad senseand refer to the orderly or controlled form of cell death in mammalsthat is typically accompanied by one or more characteristic cellchanges, including condensation of cytoplasm, loss of plasma membranemicrovilli, segmentation of the nucleus, degradation of chromosomal DNAor loss of mitochondrial function. This activity can be determined andmeasured, for instance, by cell viability assays (such as Alamar blueassays or MTT assays), FACS analysis, caspase activation, DNAfragmentation (see, for example, Nicoletti et al., J. Immunol. Methods,139:271-279 (1991), and poly-ADP ribose polymerase, “PARP”, cleavageassays known in the art.

As used herein, the term “disorder” in general refers to any conditionthat would benefit from treatment with the compositions describedherein, including any disease or disorder that can be treated byeffective amounts of polypeptides such as Apo2L/TRAIL. This includeschronic and acute disorders, as well as those pathological conditionswhich predispose the mammal to the disorder in question. Non-limitingexamples of disorders to be treated herein include benign and malignantcancers; inflammatory, angiogenic, and immunologic disorders, autoimmunedisorders, arthritis (including rheumatoid arthritis), multiplesclerosis, and HIV/AIDS.

The terms “cancer”, “cancerous”, or “malignant” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, leukemia, blastoma, and sarcoma. Moreparticular examples of such cancers include squamous cell carcinoma,myeloma, small-cell lung cancer, non-small cell lung cancer, glioma,gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer,lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer,endometrial cancer, kidney cancer, prostate cancer, thyroid cancer,neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervicalcancer, stomach cancer, bladder cancer, hepatoma, breast cancer, coloncarcinoma, and head and neck cancer. Optionally, the cancer cellsexpress DR4 and/or DR5 receptor(s).

The terms “treating”, “treatment” and “therapy” as used herein refer tocurative therapy, prophylactic therapy, and preventative therapy.Consecutive treatment or administration refers to treatment on at leasta daily basis without interruption in treatment by one or more days.Intermittent treatment or administration, or treatment or administrationin an intermittent fashion, refers to treatment that is not consecutive,but rather cyclic in nature.

The term “mammal” as used herein refers to any mammal classified as amammal, including humans, cows, horses, dogs and cats. In a preferredembodiment of the invention, the mammal is a human.

B. Exemplary Methods and Materials for Carrying Out the Invention

The present invention provides various formulations, and methods formaking such formulations, of Apo2L/TRAIL. Various formulation excipientscan enhance solubility of Apo2L/TRAIL in formulations, for instance,which are acceptable for pharmaceutical uses and/or enhance stability ofthe Apo2L/TRAIL protein in a form (e.g., trimer form) which hasbiological activity. For example, Applicants have found that thepresence of various excipients (for instance, arginine salts) in suchformulations can markedly increase the solubility and stability ofApo2L/TRAIL.

The unexpected finding of the readily reversible crystallization ofApo2L/TRAIL further provides basis for purification methods and stableformulations of Apo2L/TRAIL. In particular, forming crystals andsubsequently drying the material by various methods (includinglyophilization) may provide long term stability of bulk preparations ofthe protein. Further, lyophilized crystal compositions are expected toretain stability through a range of temperatures. Applicants have foundthat a preferred water content of the dried crystals (useful foroptimizing trimer stability) is at least about 5% water, and preferablyabout 5% to about 10% water. Particularly, Applicants found that forstorage at temperatures greater than 30° C., reducing the water contentof the dried crystals to below about 5% water may adversely affect thestorage-stability of the Apo2L/TRAIL protein crystals. Addition of oneor more excipients, such as sucrose, trehalose, arginine, or Captisol™to the protein crystal compositions may impove or enhancestorage-stability of the protein crystals.

Dried crystals can also be employed in suspension formulations suitablefor, e.g., subcutaneous or intramuscular administration. As described inthe Examples, sodium salts, particularly sodium sulfate (Na₂SO₄),provided ready and reversible crystallization, with retention ofbiological activity upon re-dissolution of the protein. The proteincrystals then readily re-dissolved in water or an aqueous buffer, e.g. acarboxylic acid salt of an amino acid, or can be suspended innon-aqueous media without loss in physicochemical properties that can beimportant for the protein's biological activity.

Generally, the formulations are prepared using Apo2L/TRAIL polypeptides(proteins), at the desired degree of purity, and various excipients orcomponents, described below.

Production of Apo2L/TRAIL

The description below relates to methods of producing Apo2L/TRAIL byculturing host cells transformed or transfected with a vector containingApo2L/TRAIL encoding nucleic acid and recovering the polypeptide fromthe cell culture.

The DNA encoding Apo2L/TRAIL may be obtained from any cDNA libraryprepared from tissue believed to possess the Apo2L/TRAIL mRNA and toexpress it at a detectable level. Accordingly, human Apo2L/TRAIL DNA canbe conveniently obtained from a cDNA library prepared from humantissues, such as the bacteriophage library of human placental cDNA asdescribed in PCT Publication WO97/25428. The Apo2L/TRAIL-encoding genemay also be obtained from a genomic library or by oligonucleotidesynthesis.

Libraries can be screened with probes (such as antibodies to theApo2L/TRAIL or oligonucleotides of at least about 20-80 bases) designedto identify the gene of interest or the protein encoded by it. Screeningthe cDNA or genomic library with the selected probe may be conductedusing standard procedures (Sambrook et al., Molecular Cloning: ALaboratory Manual; New York: Cold Spring Harbor Laboratory Press, 1989).An alternative means to isolate the gene encoding Apo2L/TRAIL is to usePCR methodology (Sambrook et al., supra; Dieffenbach et al., PCR Primer:A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1995).

Amino acid sequence fragments or variants of Apo2L/TRAIL can be preparedby introducing appropriate nucleotide changes into the Apo2L/TRAIL DNA,or by synthesis of the desired Apo2L/TRAIL polypeptide. Such fragmentsor variants represent insertions, substitutions, and/or deletions ofresidues within or at one or both of the ends of the intracellularregion, the transmembrane region, or the extracellular region, or of theamino acid sequence shown for the full-length Apo2L/TRAIL in FIG. 1 (SEQID NO:1). Any combination of insertion, substitution, and/or deletioncan be made to arrive at the final construct, provided that the finalconstruct possesses, for instance, a desired biological activity orapoptotic activity as defined herein. In a preferred embodiment, thefragments or variants have at least about 80% amino acid sequenceidentity, more preferably, at least about 90% sequence identity, andeven more preferably, at least 95%, 96%, 97%, 98% or 99% sequenceidentity with, for example, the sequences identified herein for theintracellular, transmembrane, or extracellular domains of Apo2L/TRAIL,or the full-length sequence for Apo-2L/TRAIL. The amino acid changesalso may alter post-translational processes of the Apo-2L/TRAIL, such aschanging the number or position of glycosylation sites or altering themembrane anchoring characteristics.

Variations in the Apo2L/TRAIL sequence as described above can be madeusing any of the techniques and guidelines for conservative andnon-conservative mutations set forth in U.S. Pat. No. 5,364,934. Theseinclude oligonucleotide-mediated (site-directed) mutagenesis, alaninescanning, and PCR mutagenesis.

Scanning amino acid analysis can be employed to identify one or moreamino acids along a contiguous sequence. Among the preferred scanningamino acids are relatively small, neutral amino acids. Such amino acidsinclude alanine, glycine, serine and cysteine. Alanine is typically apreferred scanning amino acid among this group because it eliminates theside-chain beyond the beta-carbon and is less likely to alter themain-chain conformation of the variant. (Cunningham et al., Science1989, 244:1081). Alanine is also typically preferred because it is themost common amino acid. Further, it is frequently found in both buriedand exposed positions (Creighton, The Proteins, (W.H. Freeman & Co.,NY); Chothia, J. Mol. Biol. 1976, 150:1).

Particular Apo2L/TRAIL variants of the present invention include thoseApo2L/TRAIL polypeptides which include one or more of the recitedalanine substitutions provided in TABLE I of published PCT applicationWO 01/00832. Such Apo2L/TRAIL variants will typically comprise anon-naturally occurring amino acid sequence which differs from a nativeApo2L/TRAIL amino acid sequence (such as provided in FIG. 1; SEQ IDNO:1, for a full length or mature form of Apo2L/TRAIL or anextracellular domain sequence thereof) in at least one or more aminoacids. Optionally, the one or more amino acids which differ in theApo2L/TRAIL variant as compared to a native Apo2L/TRAIL will compriseamino acid substitution(s) such as those indicated in Table I of WO01/00832. Apo2L/TRAIL variants of the invention include solubleApo2L/TRAIL variants comprising residues 91-281, 92-281, 95-281 or114-281 of FIG. 1 (SEQ ID NO:1) and having one or more amino acidsubstitutions. Preferred Apo2L/TRAIL variants will include thosevariants comprising residues 91-281, 92-281, 95-281 or 114-281 of FIG. 1(SEQ ID NO:1) and having one or more amino acid substitutions whichenhance biological activity, such as receptor binding. A particularlypreferred variant comprises residues 114-281 of FIG. 1 (SEQ ID NO:1). Ina specific embodiment, Apo-2L/TRAIL consists of residues 114-281 of FIG.1 (SEQ ID NO:1).

As described in WO 01/00832 published Jan. 4, 2001, the x-ray crystalstructure of the extracellular domain of Apo2L/TRAIL was identified, andalanine-scanning mutagenesis was performed to provide the mapping of itsreceptor contact regions. The structure obtained for Apo2L/TRAILrevealed a homotrimeric protein which contains a novel divalent metalion (zinc) binding site that coordinates the interaction of theApo2L/TRAIL trimer molecule's three subunits. Like other members of theTNF family, Apo2L/TRAIL appears to comprise a compact trimer formed ofthree jelly roll monomers which bury approximately 5100 Angstrom² (1700Angstrom² per monomer) to form the globular trimer. The position of thecore beta-strands was well conserved compared to the other structurallycharacterized members of the TNF family, TNF-alpha, TNF-beta, and CD40Lwhen compared to the core strands of TNF-alpha or TNF-beta.

Variations in the Apo2L/TRAIL sequence also included within the scope ofthe invention relate to amino-terminal derivatives or modified forms.Such Apo2L/TRAIL sequences may include any of the Apo2L/TRAILpolypeptides described herein having a methionine or modified methionine(such as formyl methionyl or other blocked methionyl species) at theN-terminus of the polypeptide sequence.

The nucleic acid (e.g., cDNA or genomic DNA) encoding native or variantApo2L/TRAIL may be inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. Various vectors arepublicly available. The vector components generally include, but are notlimited to, one or more of the following: a signal sequence, an originof replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence, each of which isdescribed below. Optional signal sequences, origins of replication,marker genes, enhancer elements and transcription terminator sequencesthat may be employed are known in the art and described in furtherdetail in PCT Publication WO97/25428.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to theApo2L/TRAIL nucleic acid sequence. Promoters are untranslated sequenceslocated upstream (5′) to the start codon of a structural gene (generallywithin about 100 to 1000 bp) that control the transcription andtranslation of a particular nucleic acid sequence, such as theApo2L/TRAIL nucleic acid sequence, to which they are operably linked.Such promoters typically fall into two classes, inducible andconstitutive. Inducible promoters are promoters that initiate increasedlevels of transcription from DNA under their control in response to somechange in culture conditions, e.g., the presence or absence of anutrient or a change in temperature. At this time a large number ofpromoters recognized by a variety of potential host cells are wellknown. These promoters are operably linked to Apo2L/TRAIL encoding DNAby removing the promoter from the source DNA by restriction enzymedigestion and inserting the isolated promoter sequence into the vector.Both the native Apo2L/TRAIL promoter sequence and many heterologouspromoters may be used to direct amplification and/or expression of theApo2L/TRAIL DNA.

Promoters suitable for use with prokaryotic and eukaryotic hosts areknown in the art, and are described in further detail in PCT PublicationNo. WO97/25428.

Preferred methods for the production of soluble Apo2L/TRAIL in E. coliemploy an inducible promoter for the regulation of product expression.The use of a controllable, inducible promoter allows for culture growthto the desirable cell density before induction of product expression andaccumulation of significant amounts of product which may not be welltolerated by the host.

Three inducible promoter systems (T7 polymerase, trp and alkalinephosphatase (AP)) have been evaluated by Applicants for the expressionof Apo2L/TRAIL (amino acids 114-281). The use of each of these threepromoters resulted in significant amounts of soluble, biologicallyactive Apo2L/TRAIL trimer being recovered from the harvested cell paste.The AP promoter is preferred among these three inducible promotersystems tested because of tighter promoter control and the higher celldensity and titers reached in harvested cell paste.

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and re-ligated in theform desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures can be used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced using standard techniques known in the art.(See, e.g., Messing et al., Nucleic Acids Res. 1981, 9:309; Maxam etal., Methods in Enzymology 1980, 65:499).

Expression vectors that provide for the transient expression inmammalian cells of DNA encoding Apo2L/TRAIL may be employed. In general,transient expression involves the use of an expression vector that isable to replicate efficiently in a host cell, such that the host cellaccumulates many copies of the expression vector and, in turn,synthesizes high levels of a desired polypeptide encoded by theexpression vector (Sambrook et al., supra). Transient expressionsystems, comprising a suitable expression vector and a host cell, allowfor the convenient positive identification of polypeptides encoded bycloned DNAs, as well as for the rapid screening of such polypeptides fordesired biological or physiological properties. Thus, transientexpression systems are particularly useful in the invention for purposesof identifying analogs and variants of Apo2L/TRAIL that are biologicallyactive Apo2L/TRAIL.

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of Apo2L/TRAIL in recombinant vertebrate cell culture aredescribed in Gething et al., Nature 1981, 293:620-625; Mantei et al.,Nature 1979, 281:40-46; EP 117,060; and EP 117,058.

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes for this purpose include but are not limited to eubacteria,such as Gram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. Preferably, the host cell should secreteminimal amounts of proteolytic enzymes.

E. coli is the preferred host cell for use in the present invention. E.coli is particularly well suited for the expression of Apo2L/TRAIL(comprising amino acids 114-281 of FIG. 1), a polypeptide of under 20 kdin size with no glycosylation requirement. As a production host, E. colican be cultured to relatively high cell density and is capable ofproducing relatively high levels of heterologous proteins.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forApo2L/TRAIL-encoding vectors. Suitable host cells for the expression ofglycosylated Apo2L/TRAIL are derived from multicellular organisms.Examples of all such host cells, including CHO cells, are describedfurther in PCT Publication No. WO97/25428.

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors for Apo2L/TRAIL productionand cultured in nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Successful transfection is generallyrecognized when any indication of the operation of this vector occurswithin the host cell.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described in Sambrook et al., supra, orelectroporation is generally used for prokaryotes or other cells thatcontain substantial cell-wall barriers. Infection with Agrobacteriumtumefaciens is used for transformation of certain plant cells, asdescribed (Shaw et al., Gene 1983, 23:315 and PCT Publication No. WO89/05859). In addition, plants may be transfected using ultrasoundtreatment, PCT Publication No. WO 91/00358 published 10 Jan. 1991.

For mammalian cells without such cell walls, the calcium phosphateprecipitation method (Graham and van der Eb, Virology 1978, 52:456-457)may be employed. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact. 1977, 130:946 and Hsiao et al.Proc. Natl. Acad. Sci. USA 1979, 76:3829. However, other methods forintroducing DNA into cells, such as by nuclear microinjection,electroporation, bacterial protoplast fusion with intact cells, orpolycations, e.g., polybrene, polyornithine, may also be used. Forvarious techniques for transforming mammalian cells, see Keown et al.Methods in Enzymology 1990, 185:527-537 and Mansour et al. Nature 1988,336:348-352.

Prokaryotic cells used to produce Apo2L/TRAIL may be cultured insuitable culture media as described generally in Sambrook et al., supra.Particular forms of culture media that may be employed for culturing E.coli are described further in PCT application WO 01/00832. Mammalianhost cells used to produce Apo2L/TRAIL may be cultured in a variety ofculture media.

Examples of commercially available culture media include Ham's F10(Sigma), Minimal Essential Medium (“MEM”, Sigma), RPMI-1640 (Sigma), andDulbecco's Modified Eagle's Medium (“DMEM”, Sigma). Any such media maybe supplemented as necessary with hormones and/or other growth factors(such as insulin, transferrin, or epidermal growth factor), salts (suchas sodium chloride, calcium, magnesium, and phosphate), buffers (such asHEPES), nucleosides (such as adenosine and thymidine), antibiotics (suchas Gentamycin™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

In general, principles, protocols, and practical techniques formaximizing the productivity of mammalian cell cultures can be found inMammalian Cell Biotechnology: A Practical Approach, M. Butler, ed. (IRLPress, 1991).

Expression of the Apo2L/TRAIL may be measured in a sample directly, forexample, by conventional Southern blotting, Northern blotting toquantitate the transcription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA1980, 77:5201-5205), dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Various labels may be employed, most commonlyradioisotopes, and particularly ³²P. However, other techniques may alsobe employed, such as using biotin-modified nucleotides for introductioninto a polynucleotide. The biotin then serves as the site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionucleotides, fluorescers or enzymes. Alternatively,antibodies may be employed that can recognize specific duplexes,including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes orDNA-protein duplexes. The antibodies in turn may be labeled and theassay may be carried out where the duplex is bound to a surface, so thatupon the formation of duplex on the surface, the presence of antibodybound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. With immunohistochemicalstaining techniques, a cell sample is prepared, typically by dehydrationand fixation, followed by reaction with labeled antibodies specific forthe gene product coupled, where the labels are usually visuallydetectable, such as enzymatic labels, fluorescent labels, luminescentlabels, and the like.

Antibodies useful for immunohistochemical staining and/or assay ofsample fluids may be either monoclonal or polyclonal, and may beprepared in any mammal. Conveniently, the antibodies may be preparedagainst a native Apo2L/TRAIL polypeptide or against a synthetic peptidebased on the DNA sequences provided herein or against exogenous sequencefused to Apo2L/TRAIL DNA and encoding a specific antibody epitope.

Apo2L/TRAIL preferably is recovered from the culture medium as asecreted polypeptide, although it also may be recovered from host celllysates when directly produced without a secretory signal. If theApo2L/TRAIL is membrane-bound, it can be released from the membraneusing a suitable detergent solution (e.g., Triton-X 100) or itsextracellular region may be released by enzymatic cleavage.

When Apo2L/TRAIL is produced in a recombinant cell other than one ofhuman origin, the Apo2L/TRAIL is free of proteins or polypeptides ofhuman origin. However, it is usually necessary to recover or purifyApo2L/TRAIL from recombinant cell proteins or polypeptides to obtainpreparations that are substantially homogeneous as to Apo2L/TRAIL. As afirst step, the culture medium or lysate may be centrifuged to removeparticulate cell debris. Apo2L/TRAIL thereafter is purified fromcontaminant soluble proteins and polypeptides, with the followingprocedures being exemplary of suitable purification procedures: byfractionation on an ion-exchange column such as SP-sepharose orCM-sepharose; hydroxyapatite; hydrophobic interaction chromatography;ethanol precipitation; chromatofocusing; ammonium sulfate precipitation;gel filtration using, for example, Sephadex G-75; and diafiltration.

The Apo2L/TRAIL can be isolated by affinity chromatography. Apo2L/TRAILfragments or variants in which residues have been deleted, inserted, orsubstituted are recovered in the same fashion as native Apo2L/TRAIL,taking account of any substantial changes in properties occasioned bythe variation. For example, preparation of an Apo2L/TRAIL fusion withanother protein or polypeptide, e.g., a bacterial or viral antigen,facilitates purification; an immunoaffinity column-containing antibodyto the antigen can be used to adsorb the fusion polypeptide.

A protease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF) alsomay be useful to inhibit proteolytic degradation during purification,and antibiotics may be included to prevent the growth of adventitiouscontaminants. One skilled in the art will appreciate that purificationmethods suitable for native Apo2L/TRAIL may require modification toaccount for changes in the character of Apo2L/TRAIL or its variants uponexpression in recombinant cell culture.

During any such purification steps, it may be desirable to expose therecovered Apo2L/TRAIL to a divalent metal ion-containing solution or topurification material (such as a chromatography medium or support)containing one or more divalent metal ions. The divalent metal ionsand/or reducing agent may be used during recovery or purification of theApo2L/TRAIL. Optionally, both divalent metal ions and reducing agent,such as DTT or BME, may be used during recovery or purification of theApo2L/TRAIL. It is believed that use of divalent metal ions duringrecovery or purification will assist in providing stability ofApo2L/TRAIL trimer or preserve Apo2L/TRAIL trimer formed during the cellculturing step.

Preparation of Formulations

In the preparation of the formulations herein, it is noted that therecommended quality or “grade” of the components employed will depend onthe ultimate use of the formulation. For therapeutic uses, it ispreferred that the component(s) are of an allowable grade (such as“GRAS”) as an additive to pharmaceutical products.

In certain embodiments, there are provided compositions comprisingApo2L/TRAIL and one or more excipients which provide sufficient ionicstrength to enhance solubility and/or stability of the Apo2L/TRAIL,wherein the composition has a pH of 6 (or about 6) to 9 (or about 9).The Apo2L/TRAIL protein may be prepared by any suitable method toachieve the desired purity of the protein, for example, according to theabove methods. In preferred embodiments, the Apo2L/TRAIL proteincomprises amino acids 114-281 of FIG. 1, and more preferably, theApo2L/TRAIL protein is recombinantly expressed in E. coli host cells.The concentration of the Apo2L/TRAIL protein in the formulation may varydepending, for instance, on the intended use of the formulation. Thoseskilled in the art can determine without undue experimentation thedesired concentration of the Apo2L/TRAIL protein. For therapeutic uses,the concentration of the Apo2L/TRAIL protein in the formulation isoptionally about 0.1 to about 100 mg/ml, about 1 to about 20 mg/ml,about 10 to about 20 mg/ml, or about 20 mg/ml.

The one or more excipients in the formulations which provide sufficientionic strength to enhance solubility and/or stability of the Apo2L/TRAILis optionally a polyionic organic or inorganic acid, aspartate, sodiumsulfate, sodium succinate, sodium acetate, sodium chloride, Captisol™,Tris, arginine salt or other amino acids, sugars and polyols such astrehalose and sucrose. Preferably the one or more excipients in theformulations which provide sufficient ionic strength is a salt. Saltswhich may be employed include but are not limited to sodium salts andarginine salts. The type of salt employed and the concentration of thesalt is preferably such that the formulation has a relatively high ionicstrength which allows the Apo2L/TRAIL in the formulation to be stable(i.e., reduce precipitation and enhance trimer content) and/or whichallows the soluble protein concentration to exceed 2 mg/ml, morepreferably, exceed 5 mg/ml, even more preferably exceed 10 mg/ml, andmost preferably to achieve a concentration of at least about 20 mg/ml.Optionally, the salt is present in the formulation at a concentration ofabout 20 mM to about 0.5 M. In more preferred embodiments, the salt isan arginine salt or sodium sulfate. Optionally, the arginine salt maycomprise arginine citrate, arginine tartrate, arginine malate, argininesuccinate, arginine phosphate, and arginine sulfate. More preferably,the arginine salt is present in a concentration of about 0.2 M to about0.5 M. It is noted that while arginine tartrate is useful as anexcipient in the formulations described herein, the use of tartaric acidas a vehicle at higher concentrations (such as hundreds of mM) may notbe desirable for parenteral administration or human clinicalapplications. Applicants have observed in an in vivo animal study thatvehicle concentrations of 0.5M arginine neutralized with 0.25M tartrateadministered intravenously at greater than 5 ml/kg/hr can have adeleterious effect on renal tissue. Accordingly, there may be an upperthreshold concentration of tartaric acid beyond which one skilled in theart would not select for clinical, therapeutic uses.

If relatively lower concentrations of Apo2L/TRAIL protein are desired inthe formulation, for instance, less than about 5 mg/ml, or less thanabout 2 mg/ml, or about 0.1 to about 2 mg/ml, the excipient providingstability to the formulation may be a sugar, such as trehalose, sucrose,glucose, lactitol, or lactose. Optionally, the sugar may be employed inthe formulations at a concentration of about 1% to about 8%. Theexcipient may also be an arginine salt, as described above.

The composition preferably has a pH of 6 (or about 6) to 9 (or about 9),more preferably about 6.5 to about 8.5, and even more preferably about 7to about 7.5. In a preferred aspect of this embodiment, the compositionwill further comprise a buffer to maintain the pH of the composition atleast about 6 to about 8. Examples of buffers which may be employedinclude but are not limited to Tris, HEPES, and histidine. Whenemploying Tris, the pH may optionally be adjusted to about 7 to 8.5.When employing Hepes or histidine, the pH may optionally be adjusted toabout 6.5 to 7. Optionally, the buffer is employed at a concentration ofabout 5 mM to about 50 mM in the formulation, and preferably at aconcentration of about 10 mM to about 20 mM.

Particularly for liquid formulations (or reconstituted lyophilizedformulations), it may be desirable to include one or more surfactants inthe composition. Such surfactants may, for instance, comprise anon-ionic surfactant like TWEEN™ or PLURONICS™ (e.g., polysorbate orpoloxamer). Preferably, the surfactant comprises polysorbate 20 (“Tween20”). The surfactant will optionally be employed at a concentration ofabout 0.005% to about 0.2%.

Preferred formulations are those in which the excipient(s) provide foroptimized Apo2L/TRAIL trimer content and minimize the amount ofApo2L/TRAIL dimer formation (or aggregate formation) or changes incharge distribution. Optionally, the formulation contains no more than5% Apo2L/TRAIL aggregates, no more than 10% Apo2L/TRAIL disulfide linkeddimer (of the total amount of Apo2L/TRAIL protein in the formulation),and/or no more than 10% change in the initial charge distribution.

In preferred embodiments, the present invention provides compositionscomprising about 0.1 mg/ml to about 20 mg/ml of Apo2L/TRAIL and anarginine salt, wherein the composition has a pH of about 6.5 to about8.5 (optionally, 6.8 to 7.5). Optionally, the compositions furthercomprise a buffer such as Tris and/or a surfactant such as polysorbate20. Preferably, the Apo2L/TRAIL protein does not include (i.e., is notlinked or fused to) any epitope tag molecule(s) or leucine zippermolecule(s).

In even more preferred embodiments, the present invention providescompositions comprising about 2 to about 20 mg/ml of Apo2L/TRAIL, about0.4 to about 0.5M arginine salt, and buffer, wherein the composition hasa pH of about 6.5 to about 7.5.

Optionally, a divalent metal ion may be included in the formulations.The divalent metal ion may be a zinc molecule, such as zinc sulfate,zinc chloride, or zinc acetate. The divalent metal ion can optionally beincluded in the formulation at a concentration of about 50 micromolar toabout 400 micromolar.

The formulations of the present invention may include, in addition toApo2L/TRAIL and those components described above, further various otherexcipients or components. Optionally, the formulation may contain, forparenteral administration, a pharmaceutically or parenterally acceptablecarrier, i.e., one that is non-toxic to recipients at the dosages andconcentrations employed and is compatible with other ingredients of theformulation. Optionally, the carrier is a parenteral carrier, such as asolution that is isotonic with the blood of the recipient. Examples ofsuch carrier vehicles include water, saline or a buffered solution suchas phosphate-buffered saline (PBS), Ringer's solution, and dextrosesolution. Various optional pharmaceutically acceptable carriers,excipients, or stabilizers are described further in Remington'sPharmaceutical Sciences, 16th edition, Osol, A. ed. (1980).

The formulations herein also may contain one or more preservatives.Examples include octadecyldimethylbenzyl ammonium chloride,hexamethonium chloride, benzalkonium chloride (a mixture ofalkylbenzyldimethylammonium chlorides in which the alkyl groups arelong-chain compounds), and benzethonium chloride. Other types ofpreservatives include aromatic alcohols, alkyl parabens such as methylor propyl paraben, and m-cresol. Antioxidants include ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; butyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; sugars such as sucrose, mannitol,trehalose or sorbitol; or polyethylene glycol (PEG).

Additional examples of such carriers include lecithin, serum proteins,such as human serum albumin, buffer substances such as glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts, or electrolytes such as protaminesulfate, sodium chloride, polyvinyl pyrrolidone, and cellulose-basedsubstances. Carriers for gel-based forms include polysaccharides such assodium carboxymethylcellulose or methylcellulose, polyvinylpyrrolidone,polyacrylates, polyoxyethylene-polyoxypropylene-block polymers,polyethylene glycol, and wood wax alcohols. Conventional depot formsinclude, for example, microcapsules, nano-capsules, liposomes, plasters,inhalation forms, nose sprays, and sustained-release preparations.

The compositions of the invention may comprise liquid formulations(liquid solutions or liquid suspensions), and lyophilized formulations,as well as suspension formulations in which the Apo2L/TRAIL protein isin the form of crystals or amorphous precipitate.

The final formulation, if a liquid, is preferably stored frozen at ≦20°C. Alternatively, the formulation can be lyophilized and provided as apowder for reconstitution with water for injection that optionally maybe stored at 2-30° C.

The formulation to be used for therapeutic administration must besterile. Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticcompositions generally are placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

The composition ordinarily will be stored in single unit or multi-dosecontainers, for example, sealed ampules or vials, as an aqueous solutionor as a lyophilized formulation for reconstitution. The containers mayany available containers in the art and filled using conventionalmethods. Optionally, the formulation may be included in an injection pendevice (or a cartridge which fits into a pen device), such as thoseavailable in the art (see, e.g., U.S. Pat. No. 5,370,629), which aresuitable for therapeutic delivery of the formulation. As an example of alyophilized formulation, 10 mL vials are filled with 5.5 mL ofsterile-filtered 2% (w/v) aqueous Apo2L/TRAIL solution, and theresulting mixture is lyophilized. An injection solution can be preparedby reconstituting the lyophilized Apo2L/TRAIL formulation using, forexample, Water-for-Injection.

In further more particular embodiments of the formulations, there areprovided compositions which include Apo2L/TRAIL crystals. For instance,the composition may comprise a suspension formulation comprisingApo2L/TRAIL crystals. Applicants surprisingly found that the solid stateof Apo2L/TRAIL protein at 5° C. is crystalline at moderate to low ionicconditions, unlike many other proteins known in the art that are solubleor form amorphous precipitates under similar conditions. Further, it wasfound that the solid state of the Apo2L/TRAIL crystals reversiblysolubilizes when brought to ambient temperature (i.e., room temperature)without a loss in protein biological activity or adverse effect on thebiochemical properties of the protein. This observation was quitedifferent from the denaturation or irreversible precipitation observedfor other proteins known in the art.

Optionally, the Apo2L/TRAIL crystals are prepared by cooling asuper-saturated solution of Apo-2L/TRAIL protein from about 20 to about30° C. to below about 15° C., preferably about 2 to 8° C., and morepreferably, below about 2-8° C. Crystallization can be carried out inbatch or semi-batch mode at a large range of scale, from a fewmilliliters to hundreds of liters of solution. The crystallization ratecan be controlled by programmed cooling and agitation. The equipment mayinclude, but is not limited to, agitated or static tanks with surfaceand/or internal temperature control. Internal baffles and draft tubesmay also be used to enhance mixing in agitated tanks. Crystal nucleationcan also be controlled by seeding [Moore, AIChE Practical EngineeringPerspectives, Distillation and Other Industrial Separations, pp.239-245]. The degree of super-saturation, salt composition, coolingrate, agitation rate, and seeding can affect crystal formation rate,crystal size distribution, and crystal yield.

The Apo2L/TRAIL crystal slurry may be washed to remove the salts.Optionally, the crystal slurry may be washed with water. Alternatively,the crystal slurry may be equilibrated to a low ionic strength.Subsequently, the material may be dried for storage or preparation forparenteral formulations. The crystal drying methods may include but arenot limited to static vacuum drying, vacuum drying with vibration,rotation, or agitation motion facilitated by dry air/N2 flow,lyophilization, spray drying and fluidized bed drying.

Applicants have found that it may be desirable to achieve a watercontent of at least about 5%, and preferably about 5% to about 10% inthe dried crystals. It will be within the ordinary skill in the art forthe artisan to control the water content of the crystal compositionusing known techniques, such as by stopping the drying (e.g.,lyophilization) process(s) at designated time points or by drying thecrystal compositon completely (or until minimal water is present) andthen adding back controlled amounts of water to achieve the desiredamount of re-hydration to the protein crystals.

Optionally, to prepare the crystals, the solution of Apo-2L/TRAILprotein contains sodium sulphate or sodium chloride. Optionally, thesalt concentration is about 25 mM to about 150 mM and optionally the pHis about 6 to about 9 (preferably, pH of about 6.5 to about 8.5).

Optionally, other excipients, such as sucrose, trehalose, glucose,lactitol, lactose, arginine, or Captisol™, may be included in thecrystal preparation. Optionally, the molar ratio of such excipient toApo2L/TRAIL trimer is about 1 millimole to about 300 moles excipient tomoles of Apo2L/TRAIL trimer. Optionally, the sugar may be employed inthe crystal preparation at a concentration of about 0.001% (w/w) toabout 65% (w/w). Such excipients may optionally be added to the wetcrystal slurry (prior to drying) so that the excipient(s) can diffuseinto the crystalline composition.

The Apo2L/TRAIL crystals can be reconstituted to a liquid formulationand sterilized for parenteral injection. Alternatively, the driedcrystals can be suspended in a high viscosity biocompatible medium forsubcutaneous or intramuscular administration. The suspending medium maybe aqueous or non-aqueous. Examples of aqueous suspensions includecellulose-based systems such as carboxymethyl cellulose,hydroxyethylcellulose, or polymer-based systems like polylacticacid-glycolic acid (PGLA). An example of non-aqueous medium is sucroseacetate isobutyrate (SAIB) predissolved in solvents such as ethanol,propylene carbonate, or N-methylpyrrolidone. A suspension of uniformsize distribution can be prepared by homogenizing the dried crystals inthe viscous medium using, by way of example, a probe homogenizer or amicrofluidizer.

Methods of Use and Other Applications

In one embodiment of the invention, there is provided an improved methodfor purifying and storing Apo2L/TRAIL protein. More particularly, themethods of purification employ crystallization of Apo2L/TRAIL and thecrystals can be dried for storage. The methods provide an effective,efficient, and cost saving alternative to, for instance, purificationprotocols requiring multiple column purifications. Drying thecrystalline material can also provide a relatively low volume, effectiveway of bulk storage which avoids freezing the purified material in bulkcontainers and thawing the frozen bulk material.

In the methods, an Apo2L/TRAIL preparation, such as a cell pastecontaining recombinantly expressed Apo2L/TRAIL protein, is provided.Optionally, though not required, the cell paste may be processed (forinstance, may be exposed to one or more reducing agents such as DTT orBME) or partially purified using any suitable methods known in the art,such as cation exchange chromatography methods. Cation exchangechromatography materials may optionally be SP-Sepharose, CM-Sepharose,or Macro-prep ceramic HS resin. Applicants have found that addition ofpolyethylene glycol to the pool being applied to such chromatographycolumns may assist in increasing yield of crystalline protein. Variouspolyethylene glycols are commercially available (such as from NektarCorporation). Polyethelyene glycols of various molecular weights may beemployed (such as from 100 to 10,000), preferably a PEG of approximately400 D is employed at a concentration of about 5%. It is within the skillin the art to determine optimal PEG molecular weights and concentrationsto increase protein crystal yield.

The processed or partially purified Apo2L/TRAIL in the preparation canbe crystallized from, for instance, a supersaturated solution bydecreasing temperature and agitation using the methods described herein.The crystals may then be collected, and washed with buffer (or water)(preferably a cold buffer at a temperature of about 2 to 8° C.). Thewashed crystals can be re-suspended or re-dissolved at ambienttemperature.

Re-solubilized Apo2L/TRAIL can be further purified by hydrophobicinteraction chromatography, recrystallized, washed and stored as wetcrystalline bulk material. Alternatively, the hydrophobic interaction orother chromatography step may be omitted in favor of simplyrecrystallizing. The wet crystalline bulk material can be stored at −20°C. or dried for storage at ambient temperature (room temperature) or at2-8° C. Preferably, the dried crystalline material is re-solubilized inan arginine succinate-containing formulation described above.Optionally, such a formulation can be sterile filtered and/or filled inindividual dosage vials, and lyophilized for later reconstitution orsuspension. Optionally, the dried crystalline formulation can be filledas a powder in vials and made into a solution or suspension. Asdescribed above, it may be desirable to achieve a water content of about5% to about 10% in the dried Apo2L/TRAIL crystals.

The Apo2L/TRAIL formulations described herein can be employed in avariety of therapeutic and non-therapeutic applications. Among theseapplications are methods of treating disorders, such as cancer, immunerelated conditions, or viral conditions. Such therapeutic andnon-therapeutic applications are further described, for instance, inWO97/25428, WO97/01633, and WO 01/22987.

In the methods of the invention for treating a disorder using aformulation disclosed herein, the formulation of Apo2L/TRAIL can bedirectly administered to the mammal by any suitable technique, includinginfusion or injection. The specific route of administration will depend,e.g., on the medical history of the patient, including any perceived oranticipated side effects using Apo2L/TRAIL and the particular disorderto be corrected. Examples of parenteral administration includesubcutaneous, intramuscular, intravenous, intraarterial, andintraperitoneal administration of the composition. The formulations arepreferably administered as repeated intravenous (i.v.), subcutaneous(s.c.), intramuscular (i.m.) injections or infusions, intracranialinfusions or as aerosol formulations suitable for intranasal orintrapulmonary delivery (for intrapulmonary delivery see, e.g., EP257,956).

It is noted that osmotic pressure of injections may be important insubcutaneous and intramuscular injection. Injectable solutions, whenhypotonic or hypertonic, may cause pain to a patient upon infusion.Usually, for the therapeutic, injectable formulations herein, it ispreferred that the relative osmolarity of the injectable solution beabout 300 mosm to about 600 mosm.

Apo2L/TRAIL can also be administered in the form of sustained-releasepreparations. Suitable examples of sustained-release preparationsinclude semipermeable matrices of solid hydrophobic polymers containingthe protein, which matrices are in the form of shaped articles, e.g.,films, or microcapsules. Examples of sustained-release matrices includecellulose derivatives (e.g., carboxymethylcellulose), sucrose-acetateisobutyrate (SABER™) in non-aqueous media, polyesters, hydrogels (e.g.,poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res.1981, 15: 167-277; Langer, Chem. Tech. 1982, 12: 98-105 orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919, EP 58,481),copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman etal., Biopolymers 1983, 22: 547-556), non-degradable ethylene-vinylacetate (Langer et al., supra), degradable lactic acid-glycolic acidcopolymers such as the Lupron Depot (injectable microspheres composed oflactic acid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid (EP 133,988). One optional method ofdelivery for systemic-acting drugs involves administration by continuousinfusion (using, e.g., slow-release devices or minipumps such as osmoticpumps or skin patches), or by injection (using, e.g., intravenous orsubcutaneous means, including single-bolus administration).

The composition to be used in the therapy will be formulated and dosedin a fashion consistent with good medical practice, taking into accountthe clinical condition of the individual patient, the site of deliveryof the composition, the method of administration, the scheduling ofadministration, and other factors known to practitioners. The “effectiveamounts” of each component for purposes herein are thus determined bysuch considerations and are amounts that result in bioavailability ofthe Apo2L/TRAIL or other drugs to the mammal.

As a general proposition, the total pharmaceutically effective amount ofthe Apo2L/TRAIL polypeptides administered will be in the range of fromabout 1 mg/kg/day to about 20 mg/kg/day based on kg of patient bodyweight although, as noted above, this will be subject to therapeuticdiscretion.

Although injection is preferred, an infusion device may also be employedfor continuous infusions. An intravenous bag solution may also beemployed.

It is contemplated that yet additional therapies may be employed in themethods. The one or more other therapies may include but are not limitedto, administration of radiation therapy, cytokine(s), growth inhibitoryagent(s), chemotherapeutic agent(s), cytotoxic agent(s), tyrosine kinaseinhibitors, ras farnesyl transferase inhibitors, angiogenesisinhibitors, and cyclin-dependent kinase inhibitors which are known inthe art and defined further with particularity in Section I above. Inaddition, therapies based on therapeutic antibodies that target tumorantigens such as Rituxan™ or Herceptin™ as well as anti-angiogenicantibodies such as anti-VEGF, or antibodies that target Apo2L receptors,such as DR5 or DR4.

Preparation and dosing schedules for chemotherapeutic agents may be usedaccording to manufacturers' instructions or as determined empirically bythe skilled practitioner. Preparation and dosing schedules for suchchemotherapy are also described in Chemotherapy Service Ed., M. C.Perry, Williams & Wilkins, Baltimore, Md. (1992).

It may be desirable to also administer antibodies against otherantigens, such as antibodies which bind to CD20, CD11a, CD18, CD40,ErbB2, EGFR, ErbB3, ErbB4, vascular endothelial factor (VEGF), or otherTNFR family members (such as DR4, DR5, OPG, TNFR1, TNFR2).Alternatively, or in addition, two or more antibodies binding the sameor two or more different antigens disclosed herein may beco-administered to the patient. Sometimes, it may be beneficial to alsoadminister one or more cytokines to the patient. In one embodiment, theApo2L formulations are co-administered with a growth inhibitory agent.

The Apo2L/TRAIL formulation may be administered concurrently orsequentially with such other agents. For example, the Apo2L/TRAILformulation or a chemotherapeutic agent may be administered as apre-treatment (prior to administration of any such other agents), suchas a pre-treatment of cancer cells which may otherwise be resistant tothe apoptotic effects of Apo2L/TRAIL.

The invention also provides kits which include a formulation describedherein. A typical kit will comprise a container, preferably a vial, forApo2L/TRAIL in one or more excipients as described above; andinstructions, such as a product insert or label, directing the user asto how to employ the Apo2L/TRAIL formulation. This would preferablyprovide a pharmaceutical formulation. Preferably, the pharmaceuticalformulation is for treating cancer or an immune related condition.Suitable containers include, for example, bottles, vials, syringes, andtest tubes. The containers may be formed from a variety of materialssuch as glass or plastic. The container holds an Apo2L/TRAIL formulationthat is effective for diagnosing or treating the disorder and may have asterile access port (for example, the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The label on, or associated with, the containerindicates that the formulation is used for diagnosing or treating thedisorder of choice. The article of manufacture may further comprise asecond container comprising water-for-injection, apharmaceutically-acceptable solution, saline, Ringer's solution, ordextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse.

All patents, patent applications, publications, product descriptions,and protocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties.

EXAMPLES

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va.

Formulations were prepared and assays were conducted to identifyApo2L/TRAIL formulations having desirable characteristics from atherapeutic, diagnostic, and/or commercial standpoint. In particular,Applicants sought to identify formulation components and conditionsthat, among other things, may enhance solubility of biologically activeApo2L/TRAIL, particularly at concentrations up to at least 20 mg/ml, andmay provide stability upon storage at 2-8° C. or at ambient temperature.Applicants also sought to identify Apo2L/TRAIL formulations for use inthe clinic that may preserve the protein's native non-covalent trimercontent, charge distribution, and/or biological activity during storage.

Example 1 Liquid Formulations of Apo2L/TRAIL with Enhanced Solubilityand Stability

Apo2L/TRAIL protein consisting of amino acids 114-281 (see FIG. 1) wasexpressed in E. coli under the AP promoter control (preparation andexpression described in Example 8 (Section A) of WO 01/00832 publishedJan. 4, 2001), and purified from the E. coli cell lysates by threechromatographic steps consisting of cation exchange, hydroxyapatite, andhydrophobic interaction chromatography (WO 01/00832, Example 8, SectionC). In the third chromatographic separation, the Apo2L/TRAIL protein waseluted in 600 mM Na sulfate or 400 mM ammonium sulfate, 50 mM Tris, pH7.5. The protein was then buffer exchanged to the various formulationexcipients listed in Table 1 by dialysis, and was next concentrated atambient temperature using Centricon-10 filtration up to concentrationsof 20 mg/mL. The samples were then filtered through 0.22 micron filtersand stored at either 2-8° C. or 30° C. to assess solubility andstability.

As shown in Table 1, about 20 different excipients were examined. Highpurity NF, USP, or EP grade excipients were used from common commercialsources (Sigma, Mallindkrodt) unless otherwise indicated as follows:alpha,alpha trehalose dihydrate (Pfanstiehl or Senn), sucrose(Pfanstiehl), Captisol™ (Cydex), Arginine free base (Ajinomoto or KyowaHakko Kogyo). Criteria used for initial excipient screening included 1)solubility at 2-8° C. (the storage condition of bulk preparations priorto fill in vials), 2) solubility under manufacturing scaleultrafiltration and diafiltration steps, 3) short term liquid stabilityand freeze-thaw stability, and 4) lyophilized formulation physicalstability. Solubility at 2-8° C. was evaluated by periodic visualassessment of precipitation for up to 1 month and confirmed by UVspectroscopy scan using an extinction coefficient of 1.53 at 278 nm.

Table 1 indicates that excipients having relatively high ionic strengthconditions provided solubility at concentrations of Apo2L/TRAIL above 10mg/ml at 2-8° C.

TABLE 1 Excipient Solubility At 2-8° C. 8% Trehalose <3 mg/ml 8% Sucrose<3 mg/ml 16% Sucrose <2 mg/ml 6% Lactitol <2 mg/ml 4% Sucrose/4%Mannitol <2 mg/ml 5% PEG3350 <5 mg/ml 20% Glycerol <5 mg/ml 0.15 M NaCl<5 mg/ml 0.25 M Na phosphate <5 mg/ml 0.5 M Glycine <12 mg/ml 0.5 MAspartate 10-20 mg/ml 0.5 M Na sulfate 10-20 mg/ml 0.5 M Na acetate >20mg/ml 0.5 M Na chloride >20 mg/ml 24% Sulfobutylether beta- >20 mg/mlcyclodextrin (Captisol ™) 0.5 M Tris >20 mg/ml 0.5 MArginine-tartrate >20 mg/ml

Short-term liquid stability (1 week storage time period at 30° C.) wasthen evaluated for several of those preparations shown in Table 1 thatprovided solubility of Apo2L/TRAIL at concentrations of about 20 mg/ml.The short-term stability was assessed by visual assessment of turbidity,size exclusion HPLC (SEC) to determine the amount of native trimer andaggregates in the preparations, and by ion exchange HPLC (IEX) todetermine the charge distribution. SEC was conducted using a 1) Superose12 column (Pharmacia) or 2) 4.6 mm wide by 300 mm long Toso Haas TSKSuper SW2000 column (TosoHaas) and a 13 mM Na phosphate, 400 mM ammoniumsulfate (pH 6.5) mobile phase run at a rate of 1) 0.6 ml/min ro 2) 0.15mL/min. IEX was conducted using a ProPac WCX-10 column (Dionex) at 40°C. and a NaCl gradient run at a rate of 0.5 ml/min.

The results are shown in FIG. 2. The sodium sulfate, arginine tartrate,Captisol™, and sodium acetate preparations exhibited the greateststability in one or both of the assays (FIG. 2).

Example 2 Lyophilized Apo2L/TRAIL Formulations Containing Arginine Salts

Applicants found that Apo2L/TRAIL formulations containing arginine saltscould be readily concentrated to >20 mg/mL protein by ultrafiltrationand diafiltration. Because of certain in vivo pharmacokinetic propertiesof the Apo2L/TRAIL 114-281 amino acid form of the protein (described inExample 1), Applicants particularly sought to identify a stableformulation having >20 mg/ml Apo2L/TRAIL protein.

To identify those arginine salts that provide pharmaceutically andcommercially viable lyophilized products, the physical stability ofvehicle formulations containing different arginine salts was evaluated.Arginine salts were prepared by titrating 0.5M Arginine free base withvarious acids (shown in Table 2) to give a pH 7 solution in 20 mM Tris.2 ml preparations were filled in 5 cc glass vials and subjected to aconservative long freeze-drying (lyophilization) cycle (freezing at −50°C., primary drying at 0° C. and secondary drying at 42° C.). Osmolalityof the solutions (prior to lyophilization) was also determined usingvapor pressure depression methods to identify those preparations thatmay be suitable for IV administration in a clinical setting.

Table 2 indicates that the lyophilized preparations containingpolyanionic organic or inorganic acids exhibited more desirable physicalstability than those prepared using monoanionic acids. The lyophilizedproducts of the polyanionic salts appeared as solid intact dried cakes(indicated in Table 2 as “yes”), rather than melted, gelled, collapsed,fenestrated, egg-shaped or fragmented shells (indicated in Table 2 as“no”).

In addition, 0.5M arginine salts of polyanionic acids demonstrated anosmolality which may be suitable for IV administration (less than2.5-fold hypertonic), unlike the monoionic arginine salts (e.g. 0.5Marginine-lactate gives a 3.1× hypertonic solution) and some of the otherrelatively high ionic strength preparations that exhibited goodsolubility and liquid stability (e.g., 0.5 M sodium acetate gives a 3.4fold hypertonic solution).

TABLE 2 Acceptable lyo Osmolality of 0.5 Arginine salts physicalstability? M solution* Arg-citrate Yes 505 Arg-tartrate Yes 530Arg-malate Yes 573 Arg-succinate Yes 630 Arg-oxalate Yes ND Arg-lactateNo 927 Arg-glycolate No ND Arg-acetate No 978 Arg-glutamate No 899Arg-phosphate Yes 465 Arg-sulfate Yes 462 Arg-nitrate No 774 Arg-HCL No830 *Values are mosmol/kg. Isotonic solutions have an osmolality ofapproximately 292-300. “ND” indicates value was not determined.

Based on the results obtained in the study reported in Table 2, severallyophilized Apo2L/TRAIL-containing formulations were then evaluated forbiochemical stability of the protein after storage at varioustemperatures. Apo2L/TRAIL (residues 114-281; prepared as described inExample 1) was formulated by ultrafiltration/diafiltration to 10 mg/mlin 0.5M arginine-tartrate or arginine-citrate, or to 3 mg/ml in 8%sucrose or trehalose. All of these preparations contained 20 mM Tris, pH7.0 and 0.01% polysorbate 20. The samples were then lyophilized asdescribed above. Stability was assessed by measuring content of nativetrimer and aggregates using SEC (described in Example 1) and chargedistribution using IEX (described in Example 1).

After 4 months storage at 40° C., the % trimer and % IEX main peak wasdetermined relative to an unlyophilized control solution that was storedat −70° C. (see FIG. 3A). The data in FIG. 3A indicates that thearginine salt-containing lyophilized formulations exhibited greaterstability as compared to the formulation preparations containing sucroseor trehalose.

To further examine the effects of arginine-salt type on the stability ofApo2L/TRAIL formulations, both the liquid and the lyophilizedformulations of four different arginine salt-containing 20 mg/mlApo2L/TRAIL formulations were monitored for biochemical stability of theprotein. Liquid stability was monitored at 2-8° C. and at ambienttemperatures for up to 1 month (Table 3) and lyophilized stability wasmonitored at 50° C. for one month (FIG. 3B).

In addition to conducting SEC and IEX assays described in Example 1,covalent dimer formation was monitored by SEC under denaturingconditions (SDS-SEC). The SDS-SEC assay was conducted using a TSKG2000SWXL column (TosoHaas) run at 0.6 ml/min in a mobile phaseconsisting of 25 mM sodium phosphate, 0.1% SDS, 200 mM NaCl. Sampleswere diluted to 1 mg/ml protein with a solution that gave 50 mM Tris (pH7.0), 200 mM NaCl, 0.5% SDS, pH 9 and 5 mM iodoacetamide (Sigma).Samples were then incubated at 50° C. for 10 minutes prior to HPLCanalysis.

Bioactivity of the Apo2L/TRAIL in the various formulations was alsodetermined using SK-MES-1 cells and Alamar Blue staining for viable cellcounts. In the assay, the Apo2L/TRAIL formulation (50 uL at 2 ug/mL) wasadded to assay medium (0.1% Bovine Serum Albumin, RPMI 1640) and 2-foldserial dilutions were made in 96-well plates. Then, 50 uL SK-MES-1 cells(human lung carcinoma cell line, ATCC HTB58) were added into the wellsat 20000 cells/well density. The plates were incubated for 24 hours at37° C., and Alamar Blue was added for the last 4 hours of the 24 hourincubation time. The staining intensity was determined on a fluorescenceplate reader with excitation wavelength set at 530 nm and emission at590 nm. A four-parameter fit to the data in the assay range of 0.1 to1000 ng/ml gives the ED50, or the concentration of Apo2L/TRAIL thatinduces 50% killing of the cells. Cell killing potency increases withdecreasing ED50.

As shown in Table 3, after 1 month storage of the liquid formulations at2-8° C., the four arginine-salt containing formulations showed onlysmall differences in Apo2L/TRAIL quality. The arginine-sulfateformulation exhibited the highest extent of aggregate formation. Thearginine-malate formulation exhibited the highest extent of dimerformation, and the arginine-phosphate and arginine-malate formulationsshowed the largest change in the IEX % main peak area.

After 2 weeks storage at ambient temperature, the arginine-sulfate andarginine-succinate formulations retained the highest level ofbioactivity (Table 3).

Overall, the arginine-succinate formulation demonstrated somewhatsuperior stability characteristics for Apo2L/TRAIL in the liquid state.

TABLE 3 % IEX main % peak remaining¹ % Formulations Trimer¹ (% ofcontrol) Monomer¹ % bioactivity² Arginine- 96.2 105.1 93.4 77.0 malateArginine- 96.2 94.7 94.9 88.5 succinate Arginine- 95.4 93.4 93.3 90.5sulfate Arginine- 96.0 91.0 91.8 74.3 phophate ¹Data are for 1 monthstorage at 2-8° C. ²Data are for 2 weeks storage at ambient temperature.

The stability of the lyophilized formulations containing thesearginine-salts after 1 month storage at 50° C. was also assessed by SEC,IEX and SDS-SEC (using the protocols described above). No changes in thephysical-chemical properties were observed (see FIG. 3B), suggestingthat significant stabilization of Apo2L/TRAIL may be achieved throughlyophilization of Apo2L/TRAIL formulations containing arginine-salts. AnArrhenius profile using accelerated temperature stability of alyophilized 10 mg/ml Apo2L/TRAIL formulation in 0.5M arginine-tartrate,20 mM Tris, pH 7.0, 0.01% polysorbate 20, 0.33 mM Zn sulfate is shown inFIG. 3C, and predicts a relatively long shelf life at 2-8° C., and a >2year shelf-life at ambient temper'ature. Applicants have found that alyophilized formulation (“lyo”) containing 20 mg/mL Apo2L/TRAIL in 20 mMTris, pH 7.2, 0.5 M arginine-succinate and 0.02% polysorbate 20 wasstable at temperatures as high as 50° C. for at least 12 months (Table4). The kinetics of change in IEX % main peak predicts a significantlylong (>7 year) shelf life at 2-8° C. (as well as at ambienttemperature), as with the formulation described above in FIG. 3C.

TABLE 4 % IEX main Temperature % Trimer peak % Monomer −70° C. liquid97.4 46.1 99.0 control Lyo, 2-8° C., 12 mo 97.5 46.5 99.0 Lyo, 30° C.,12 mo 97.3 45.4 98.8 Lyo, 40° C., 12 mo 97.4 44.6 98.8 Lyo, 50° C., 12mo 97.3 42.5 98.7

Example 4 Effects of pH in Apo2L/TRAIL Formulations

As described in WO 01/00832 published Jan. 4, 2001 (see also, Hymowitzet al., Biochemistry, 39:633-640 (2000), Apo2L/TRAIL protein forms ahomotrimer with a Zn-coordinated thiol at position cysteine 230 of eachmonomer, and the formation of an intramolecular disulfide bond atcysteine 230 results in loss of bioactivity of the protein.Cysteine-containing proteins are typically fOrmulated at low pH, wellbelow the pKa of the thiol groups, to prevent disulfide bond formation(see, e.g., N. Derby and T. Creighton, “Disulfide Bonds in ProteinFolding and Stability,” Methods in Enzymology, Vol 40 (Edited by B. A.Shirley; Humana Press Inc, Totowa, N.J.), Chapter 10 (1995)).Surprisingly, as the results discussed below reveal, the stability ofnative, non-covalent Apo2L/TRAIL trimer was found to decrease withdecreasing pH.

Apo2L/TRAIL consisting of amino acids 94-281 (FIG. 1) (referred toherein and in FIG. 4 as “Apo2L/TRAIL.2”) was prepared essentially asdescribed in WO 01/00832, except that a Ni-chelating affinitychromatography, instead of HIC, was used as the third chromatographicstep. Formulations were prepared by dialysis into 10 mM Na succinate (pHrange 5.5 to 6.5) or 10 mM Na phosphate (pH range of 7 to 7.5). Liquidstability of these formulations was assessed by SEC and bioactivityassays according to procedures described in the Examples above.

Within one day storage at ambient temperature, the formulations havingpH below about pH 6.5 lost trimer content and bioactivity (see FIG. 4A).This pH-stability profile was observed with other Apo2L/TRAIL proteinvariants. For example, a poly-histidine-tagged Apo2L/TRAIL formulationconsisting of amino acids 114-281 (FIG. 1) (His-Apo2L/TRAIL; preparedessentially as described by Ashkenazi et al., “Safety and AntitumorActivity of Recombinant Soluble Apo2 ligand”, JCI, 104:155-162 (1999)was prepared in 10 mM Na succinate (pH 5.5 to 6) or 10 mM Na phosphatebuffers (pH 6.5 to 7). After storage at 30° C. for 1 week, the stabilityof trimer and bioactivity (determined as described above for theformulations of FIG. 4A) was found to be enhanced at pH ≧6.5 (see FIG.4B).

Crystal structure analysis of Apo2L/TRAIL reveals that coordination ofan intrinsic Zn atom to three free thiols of cysteine 230 is needed forproper folding and native structural stability of the protein (see WO01/00832 and Hymowitz et al., supra). The unexpected pH-stabilityprofile of Apo2L/TRAIL, although not fully understood, is believed to beassociated with loss in Zn binding to the trimer as the thiol moietybecomes more protonated at lower pHs (see FIG. 5). It is believed thatneutral pH in the range of about 6.5 to about 8.5 will be most preferredfor maintaining the bioactivity and physical-chemical stability ofApo2L/TRAIL formulations.

Example 5 Effect of Surfactant on Stability of Apo2L/TRAIL

To examine the effect of surfactant on stability of Apo2L/TRAILformulations, formulations containing 20 mg/ml Apo2L/TRAIL (114-281protein (FIG. 1); prepared as described in Example 1) in 0.5 Marginine-succinate, 20 mM Tris, pH 7.2, and varying concentrations ofTween 20 (0.005%, 0.01%, 0.02%, or none (as control)) were prepared andagitated at 70 rpm and ambient temperature for up to 24 hours in glassvials positioned horizontally. Within 1 hour of agitation, an increasein light scattering (measured by absorbance in the range of 340-360 nm)was observed as the concentration of Tween 20 fell below 0.005% (FIG.6).

It is therefore believed that it may be preferable to include non-ionicsurfactant(s) such as Tween 20 in Apo2L/TRAIL formulations to enhancestabilization against agitation and handling that can denature the bulkprotein at air-water interfaces.

Example 6 Effect of Zn Sulfate on Stability of Liquid Apo2L/TRAILFormulations

A liquid formulation of Apo2L/TRAIL protein consisting of residues114-281 (FIG. 1) (prepared as described in Example 1) was prepared using20 mg/ml Apo2L/TRAIL, 0.5M arginine-tartrate, 20 mM Tris, pH 7.0, in thepresence of zero, 117 uM, or 330 uM Zn sulfate. After storage at 30° C.or 2-8° for up to 2 months, stability was evaluated by SEC, IEX, andSDS-SEC (as described above in Examples 1 and 2) relative to −70° C.control samples.

As shown in FIG. 7, addition of Zn sulfate to the formulations providedincreased stabilization against disulfide-linked intramolecular dimerformation. Though Zn sulfate did not affect the stability of Apo2L/TRAILat 2-8° C., it improved stability towards dimer formation at highertemperatures (FIG. 7).

Example 7 Reversible Crystallization of Biologically Active Apo2L/TRAIL

Applicants have found that under conditions of low Apo2L/TRAILsolubility (for example, at moderate to low ionic strength), the proteincan be crystallized. As described herein, the crystallization rate,particle size, and yield can be controlled to give useful industrialmethods for purification, bulk storage, and controlled releasesuspension formulation of Apo2L/TRAIL.

Apo2L/TRAIL protein consisting of residues 114-281 (FIG. 1) (prepared asdescribed in Example 1) was formulated at ambient temperature using NAPScolumn (Pharmacia) elution in 20 mM Tris, pH 7.2 and variousconcentrations of Na sulfate. After elution at ambient temperature,within hours, hexagonal shaped crystals of varying lengths were observed(see FIG. 8). Equilibrium solubility was reached when samples werecontinuously rotated for 3-4 days at a given temperature. There was aminimum in solubility at approximately 10-50 mM ionic strength.Solubility increased to a maximum at approximately 0.3 M Na sulfate andthen decreased until limiting solubility of the salt was reached. Thepattern was similar at higher temperatures, but solubility increasedwith increasing temperature. The observation of increasing proteinsolubility (hence decreased crystallization) with increasing saltconcentration in the hundreds of millimolar salt range is unlike commonunderstanding with respect to other proteins, where crystallizationpropensity tends to increase with increasing salt concentration (see,e.g., A. Ducruix and MM Reis-Kautt, “Solubility Diagram Analysis and theRelative Effectiveness of Different Ions on Protein Crystallization,”METHODS: A Companion to Methods in Enzymology, Vol. 1, pp. 25-30(1990)).

Monovalent cationic salts (such as sodium chloride) provided thegreatest crystallization propensity as shown in FIG. 9, while divalentcationic salts (e.g. calcium chloride and magnesium chloride)significantly reduced crystallization. Crystallization also occurred inpositively charged (lysine-salts and arginine-salts) and negativelycharged (aspartic acid) amino acid solutions below 0.3 molarconcentration (see FIG. 9), though arginine salts reducedcrystallization propensity at the same ionic strength.

To identify various crystallization process parameters, a 0.5 L solutioncontaining 5 mg/ml Apo2L/TRAIL (residues 114-281; prepared as describedin Example 1), 0.1M Na₂SO₄ and 20 mM Tris at pH 7.2 was subjected to asingle step cooling from room temperature to 5° C. Four experiments wereperformed with agitation rates ranging from 0 to 200 rpm. Thesupernatant concentration of Apo2L/TRAIL was measured in 10-minuteintervals using UV spectroscopy to monitor the progress ofcrystallization.

FIG. 10A shows that crystallization was more than 90% complete within 2hours when the bulk was agitated at 50 rpm or faster. Crystallizationwithout agitation was much slower in comparison. Static crystallizationdid not reach 90% completion until 2 days after cooling began.Crystallization rate appeared to increase with increasing agitationspeed.

FIG. 10B depicts the dissolution profile of Apo2L/TRAIL crystals underagitation. The crystal slurry was warmed from 5° C. to 30-35° C. withthe same heating rate in each dissolution experiment. Apo2L/TRAILconcentration in the supernatant was measured in 5-minute intervals tomonitor dissolution rate. The crystal dissolution rate increased whenagitation speed was increased from 50 rpm to 200 rpm. Heat transfer ratebetween the tank jacket and the crystal slurry was also enhanced whenagitation speed increased. At 100 rpm agitation rate, completedissolution was achieved within a half hour when the sample temperaturewas at approximately 35° C.

FIG. 10C shows the crystal size distribution as a function of agitationspeed during crystallization. Crystal size distribution was measuredusing a Malvern MasterSizerX Particle Size Analyzer. The mean diameter(D[v, 0.5]) decreased as agitation speed increased, and the crystal sizedistribution (D[v, 0.1] to D[v, 0.9]) became more uniform with fasteragitation. Therefore, manipulating the agitation rate duringcrystallization appears to be effective in controlling the mean diameterof the Apo2L/TRIAL crystals as well as the crystal size distribution,which may be desirable for controlled release formulations.

Example 8 Drying of Apo2L/TRAIL Crystals

To assess the feasibility of drying Apo2L/TRAIL crystals for controlledrelease formulation or bulk storage, three different drying methods wereevaluated.

The first drying method evaluated was static vacuum drying. Apo2L/TRAIL(amino acids 114-281; purified according to the method described inExample 1) was crystallized in 20 mM Tris, 0.1 M sodium sulfate, pH 7.2and washed with cold water to remove excess salt. The crystal slurry wasfilled in open glass vials and dried under 29-30 inches of mercuryvacuum at ambient temperature overnight. The dried crystals weredissolved to 2-3 mg/ml in either water or in 0.5 M arginine-salt, 20 mMTris, pH 7.2.

The second drying method evaluated was vibrational vacuum drying(apparatus obtained from SWECO Co.) to allow better flow and decreasesolid clumping. The crystal slurry was loaded on a 20 um filter toremove the bulk liquid, and the wet crystals were washed with cold Trisbuffer (20 mM, pH 7.5) or ethanol-water mixture (50%, 63%, 75%, 100%,v/v). The washed crystals were dried by passing dehumidified nitrogengas from the bottom of the filter. A slight vacuum (8-10 inches ofmercury) from the top of the filter chamber facilitated the drying rate.Furthermore, the filter chamber containing the wet crystals vibrated at1,800 rpm to break up the wet cake into fine powder during the dryingprocess. The drying process was monitored using a relative humiditysensor. Dried product was recovered through a discharge port. Theethanol-water mixture washes produced finer and better flowing powderthan crystals washed with Tris buffer, which in turn increased theprocess yield. These crystals were dissolved in a buffered 0.5M argininesalt, as described for the vacuum dried crystals.

The third drying method evaluated was lyophilization. In this method,the crystal slurry was washed with cold water and filled in glass vials.Excess bulk water was removed after the crystals settled. The slurry wasthen frozen to −50° C. Primary and secondary drying were carried out at−25° C. and 0° C., respectively. These crystals were better flowingpowders and dissolved readily in buffered 0.5M arginine-salts.

The protein quality in the dissolved crystals was then assessed usingSEC, SDS-SEC, IEX, and bioactivity assays as discussed in Examples 1 and2. Table 5 and FIGS. 11A-11B show that Apo2L/TRAIL crystals dried withthe different methods remained biochemically equivalent to thenon-crystallized frozen liquid control preparation.

TABLE 5 % trimer % monomer % IEX main Drying method by SEC by SDS-SECpeak Control (frozen starting 98.0 96.8* 53.6 material) Vibrationalvacuum drying - 97.4 98.1 54.9 water wash Vibrational vacuum drying -97.7 97.5 53.7 50% ethanol wash Vibrational vacuum drying - 97.9 97.553.4 62.5% ethanol wash Vibrational vacuum drying - 97.7 97.7 52.8 75%ethanol wash Vibrational vacuum drying - 97.8 98.0 53.3 100% ethanolwash *The apparent lower % monomer in control sample by SDS-SEC is dueto impurities in the starting material which co-elute with Apo2L dimeron SDS-SEC.

The data suggest that crystallization of Apo2L/TRAIL and subsequentdrying of the material does not adversely affect protein structure orfunction.

Example 9 Lyophilized Formulation of Crystalline-containing Apo2L/TRAILin Sodium Sulfate

To assess storage stability of crystal-containing Apo2L/TRAIL,lyophilized formulations were prepared with crystalline Apo2L/TRAIL(residues 114-281) in 20 mM Tris, pH 7-7.5, 0.2-0.5M sodium sulfate, and0.01-0.05% Tween 20. The samples were stored at various temperatures forup to 4 months. After reconstituting with sterile water, theformulations were tested for physical-chemical stability using SEC, IEX,and SDS-SEC assays described in Examples 1 and 2. Table 6 summarizes thedata for a 20 mg/ml Apo2L/TRAIL formulation in 0.2 M Na sulfate, 20 mMTris, pH 7.2, 0.01% Tween 20 after 3 months storage.

TABLE 6 % IEX main Temperature % Trimer peak % Monomer −70° C. liquidcontrol 99.3 56.1 99.1 Lyo, 2-8° C., 3 mo 99.1 58.2 99.3 Lyo, 30° C., 3mo 97.0 56.4 98.3 Lyo, 40° C., 3 mo 94.1 52.9 95.9 Lyo, 50° C., 3 mo90.1 44.8 91.7

Assuming a pseudo-first order degradation kinetics, Arrhenius profilespredict significantly longer than 2 years shelf-life for thisformulation at 2-8° C. (see FIG. 12). These preparations, though filledas clear liquid solutions of Apo2L/TRAIL, crystallize to varying degreesduring the freezing portion of the lyophilization cycle, demonstratingthat dried formulations containing crystallized Apo2L/TRAIL and sodiumsulfate have long term storage stability.

Example 10 Apo2L/TRAIL Crystallization as a Method of Recovery andPurification

The propensity of crystallization of Apo2L/TRAIL in Na sulfate solutionswas used as a means of purifying the Apo2L/TRAIL protein from E. coliextracts. The following protocol may be employed for recovery andpurification of recombinant Apo2L/TRAIL without adverse effect onprotein quality.

The harvested whole cell broth derived from E. coli (described inExample 1) was adjusted to pH 7.5 with 1.5 M Hepes (or 1.5M Tris) andthen homogenized in a homogenizer (Gaulin corporation, Everett, Mass.).The homogenate was conditioned with 5 mM DTT and flocculated with 0.1%polyethyleneimine for 1-2 hours. The flocculated material wascentrifuged by a BTPX205 (Alfa Laval Separation AB, Sweden) continuousfeed centrifuge and clarified by depth filtration. The clarified celllysate (extract) was conditioned with Triton-X100 to a finalconcentration of 0.05%. The conditioned, clarified cell lysate was thenloaded onto a cation exchange column (SP-Sepharose FF cation exchangeresin, Amersham Pharmacia, Sweden) equilibrated in 50 mM Hepes (or 50 mMTris)/0.05% Triton-X 100/1 mM DTT, pH 7.5. Apo2L/TRAIL bound to thecolumn while the non-binding proteins flowed through the column and wereremoved by washing with equilibration buffer until absorbance at 280 nmreached baseline. The column was then washed with 3 column volumes of0.1 M NaCl in equilibration buffer. The Apo2L/TRAIL was step-elutedusing 0.1 M NaCl (or 0.1M Na₂SO₄) in 50 mM each of Hepes, Tris andTriethanolamine, 0.05% Triton-X 100 and 1 mM DTT buffer, pH 7.8.

The ambient temperature Apo2L/TRAIL pool collected from the SP columnwas placed in a stainless steel tank with an insulated jacket forheating and cooling. The tank was outfitted with a conical bottom and aflush bottom valve for maximal recovery of crystallized protein. Thepool was agitated using a marine type impeller under modest mixingconditions. A temperature control skid was used to linearly ramp thetemperature from approximately 25° C. to approximately 4° C. over thecourse of 1 hour. Spontaneous crystallization was observed withinminutes after the pool reached 4° C. After more than 12 hours underthese conditions, crystallization was complete as equilibrium solubilitywas nearly established. The crystals were then captured on a filtrationassembly containing a 20 um polypropylene frit. Following crystaldeposition on the filter surface, the crystals were washed with chilled20-50 mM Tris at pH 7.5. An equal volume of wash buffer compared to theApo2L/TRAIL SP pool volume was then used to remove residual motherliquor (supernatant) from the deposited crystals. Following the wash,the crystals were dissolved in 100 mM sodium sulfate/20 mM Tris at pH7.5 by recirculating the dissolution buffer through the crystal bed atapproximately 30° C. Dissolution of the crystals was observed withinapproximately 4 hours. The dissolved, purified Apo2L/TRAIL was thensterile filtered into a container and stored frozen at −70° C.

The purity of the Apo2L/TRAIL preparations was determined by the totalE. coli protein (ECP) ELISA assays, Limulus Amebocyte Lysate (LAL)assay, and SDS-PAGE silver stain. ECP ELISA was performed byimmobilizing affinity-purified goat anti-whole ECP antibodies onmicrotiter plate wells, incubating samples and then horseradishperoxidase-conjugated ECPs. The peroxidase enzymatic activity was thenquantified with o-phenylenediamine by reading absorbance at 490 nm in amicrotiter plate reader. Endotoxin level was determined using theLimulus Amebocyte clot lysis assay. SDS-PAGE silver stain was performedon a 10 to 20% gradient polyacrylamide gel (Daiichi Pure Chemicals) inTris-glycine buffer containing 0.1% SDS. Electrophoresis was conductedat 50 mA constant current until dye front reached near the bottom of thegel. Gels were fixed and stained by Coomassie Brilliant Blue or Merrillsilver stain methods.

Protein quality was assessed by SEC, SDS-SEC, IEX, and bioactivityaccording to methods described in Examples 1 and 2.

The purity and quality of Apo2L/TRAIL recovered using the abovecrystallization method at a 60 L fermentation scale is shown in Table 7.For comparison, a reference standard purified by a three-chromatographicstep method as described in Example 1 is also shown.

TABLE 7 Protein Quality % Bio- Mono- activity % Apo2L/ Protein Purity %mer % of IEX TRAIL ECP LAL SDS- Trimer by SDS- control main Prep. (ppm)(EU/mg) PAGE by SEC SEC (±20%) peak Apo2L/ 10 0.034 No band 99.0 99.0126 63 TRAIL at purified 10 kDa by crystal- lization Refer- 0.82 0.023Band at 98.9 98.9 86 61 ence ~10 kDa material purified by standardchroma- tography

As shown in Table 7 and FIG. 13, the Apo2L/TRAIL preparation at amanufacturing scale had a high degree of purity suitable for therapeuticuse. In particular, a 10 kDa E. coli DNA binding protein that tends toco-purify with Apo2L/TRAIL was removed by the crystallization process.The data indicate that the “one-column” step purified Apo2L/TRAILprotein is amenable to crystallization and has a purity comparable to orbetter than the Apo2L/TRAIL protein purified by the three-columnpurification method described in Example 1. FIG. 14 shows the effect ofsalt type on crystallization of a one-column step purified Apo2L/TRAIL.“Poisoning” of crystallization by divalent cations was observed forpartially purified Apo2L/TRAIL (FIG. 14), similar to that observedfor >99% purified Apo2L/TRAIL shown in FIG. 9.

The biochemical properties of Apo2L/TRAIL were also not adverselyimpacted by crystallization of the partially purified Apo2L/TRAIL (seeTable 7). The data suggest that crystallization of recombinant-expressedApo2L/TRAIL, when in a partially purified state, can be an effective,efficient and cost-effective means for its purification. Optionally,such crystals can then be used for preparation of dried bulk for storageor controlled release formulations as described in Examples 8 and 9.

Example 11 Effect of Excipients on the Stability of Dried Apo2L/TRAILCrystals

Experiments were conducted to examine the effects of adding trehalose,Tween 20, Captisol™, and/or arginine to the slurry supernatant ofApo2L/TRAIL crystals, particularly the effects on the stability oflyophilized product.

Materials: 10 g Apo2L/TRAIL protein (amino acid residues 114-281 of FIG.1 prepared and purified according to the methods described in Example 1or Example 10) formulated at 10 mg/mL in 20 mM Tris, 0.1M Na₂SO₄ pH 7.2.188 mg/mL (500 mM) αα-trehalose dihydrate in 20 mM TRIS pH 7.2, 37.6mg/mL (100 mM) αα-trehalose dihydrate in 20 mM TRIS pH 7.2. 6 mg/mL (5mM) Tween 20 in 20 mM TRIS pH 7.2. 10.5 (5 mM)mg/mL Captisol™ in 20 mMTRIS pH 7.2. 4.35 mg/mL (25 mM) arginine free base, 20 mM TRISneutralized to pH 7.2 with succinic acid, 0.87 mg/mL (5 mM) argininefree base, 20 mM TRIS neutralized to pH 7.2 with succinic acid

Methods: 10 mg/mL Apo2L/TRAIL in 20 mM TRIS, 0.1M Na₂SO₄ pH 7.2 wascrystallized utilizing a single step cooling ramp as described above.After crystallization, the supernatant was removed, and the crystalswere washed three times with cold water. The crystals were re-slurriedto 100 mg/mL (final volume) and filled at 1 mL (100 mg, 1.67 umoles) per3 cc glass vial. The crystals were allowed to settle to the bottom ofthe vials, and 1 mL of the supernatant water was removed. 1 mL of eachof the following solutions was then added to each of 13 vials andswirled gently to mix the slurry.

TABLE 8 umoles Formulation Solution excipient Excipient: Apo2L #Excipient Concentration added molar ratio XF0 N/A N/A 0 0 XF1 Trehalose 188 mg/mL 500 300 XF2 Trehalose 37.6 mg/mL 100 60 XF3 Tween 20   6mg/mL 5 3 XF4 Captisol 10.5 mg/mL 5 3 XF5 Arginine 0.87 mg/mL 5 3 XF6Arginine 4.35 mg/mL 25 15

All of the vials were lyophilized as described in Example 8 andstoppered at the end of secondary drying at 0° C. under vacuum.

Three vials of each formulation were placed at 2-8° C., 30° C., 40° C.,and 50° C. One vial was analyzed for t=0 comparison. t=0 assaysincluded: TGA (thermogravimetric analysis) for moisture content, SEC foraggregation, SDS-SEC for covalent dimer formation, and IEX for chargevariants. SDS-SEC was performed both by the method described previously(Examples 1 and 2) as well as in the presence of a reducing agent(reduced SDS-SEC) to determine the extent of formation of non-thiol,covalently bonded species. Each sample was reconstituted toapproximately 20 mg/mL with 20 mM TRIS, 0.5M arginine succinate, 0.02%tween 20 pH 7.2.

The results are illustrated in FIGS. 15A-15C. Stability of thelyophilized crystal compositions was assessed by determining loss ofApo2L/TRAIL trimer by SEC and SDS-SEC on reduced and non-reducedsamples. FIG. 15A shows the results for the samples identified in TABLE8, tested at Day 0 to 24 weeks at 30° C. FIG. 15B shows the resultsafter storage of the samples at 50° C. over the course of a number ofmonths. The sample “20 mM Tris” was used as a control and consisted ofApo2L/TRAIL crystals co-lyophilized with the excipient carrier buffer(20 mM TRIS) alone. At both temperatures, the results indicate thatApo2L/TRAIL crystals having excipients such as trehalose and arginineadded prior to lyophilization have a slower rate of aggregation comparedto control. FIG. 15C illustrates the increase in % non-reducible dimerat 50° C. observed in the samples over the course of 24 weeks. Theseresults indicate that addition of excipients such as trehalose, Tween20, Captisol™, or arginine to Apo2L/TRAIL crystals prior to drying maydecrease the formation rate of this species in the protein, therebyleading to greater storage stability.

It is presently believed that the addition of one or more sugars orother excipients to crystalline forms of the Apo2L/TRAIL protein may beuseful in decreasing formation of hexameric or higher oligomeric formsof the protein.

Example 12 Effects of Water Content on Crystalline Apo2L/TRAIL

The effects of water content on Apo2L/TRAIL protein crystals wasexamined using size exclusion chromatography (SEC) and SDS-SEC, onreduced and non-reduced samples, as described in the Examples above.Apo2L/TRAIL protein was crystallized from 20 mM Tris, 0.1M Na₂SO₄, pH7.2, as described above. The crystals were washed three times with coldwater, slurried in water to a final concentration of approximately 100mg/ml, and filled at 1 mL per 3 cc vial. The crystals were lyophilizedas previously described with one third of the vials stoppered at each ofthree timepoints: end of 1° drying, part-way through 2° drying and atthe end of 2° drying, corresponding to approximately 5%, 4%, and 2%water, respectively.

The stability of the crystal samples was analyzed over the course oftime at 50° C. by measuring loss of trimer using SEC and loss of monomerto covalent dimer by both SDS-SEC and reduced SDS-SEC.

The results are shown in FIGS. 16A and 16B.

These results indicate that partial lyophilization of the Apo2L/TRAILcrystals which results in residual moisture contents greater than 2%decreases the rate of aggregate (FIG. 16A) and covalent dimer (FIG. 16B)formation of the protein.

Example 13 Effects of Water Content on Dimer and Higher AggregatedFormation in Crystalline Apo2L/TRAIL Compositions

Apo2L/TRAIL protein (prepared and purified as described in Example 10)was resolubilized in 20 mM Tris, 0.1M Na₂SO₄, pH 7.2, and crystallizedutilizing a single step temperature decrease as described above. Thecrystals were slurried in water to a final concentration ofapproximately 60 mg/ml and filled in vials at 1 ml (60 mg, 0.1micromole) per 3 cc glass vial. The crystals were lyophilized and thevials stoppered at the end of 2° drying at 0° C. under vacuum,corresponding to approximately 2% water.

Approximately 40 of the vials were opened and placed at ambienttemperature in each of four chambers having the following relativehumidities (“RH”) at 20° C.:

TABLE 9 Approximate SATURATED SALT H₂O content RH @ 20° C. SOLUTION  5%20 KC₂H₃O₂ 10% 44 K₂CO₃•2H₂O 15% 57.9 NaBr•2H₂O 20% 75.7 NaClThe vials were then stoppered and placed at 2-8°, 15°, 30°, or 50° C.

At t=0, the actual water content of the samples was determined bythermogravimetric analysis (TGA). The TGA was conducted using a TGAinstrument (Q Series™, TA Instruments, New Castle, Del.) and thetemperature of the oven was ramped to 120° C. and held until the sampleweights equilibrated.

The residual moisture content as measured by TGA is reported below inTable 10. The water content of those samples stored after 7 months at50° C. was also measured by TGA and is reported in Table 10.

TABLE 10 Relative H₂O Content H₂O (%) H₂O Content @ Humidity Expected (t= 0) Actual (t = 0) t = 7 mo, 50° C. N/A  2% 2.5 ± 0.2% 2.7 ± 0.1% 20 5% 6.7 ± 0.0% 5.7 ± 0.1% 44 10% 8.8 ± 0.2% 7.1 ± 0.0% 57.9 15% 10.4 ±0.4%  8.0 ± 0.0% 75.7 20% 11.8 ± 0.4%  9.4 ± 0.2%Several of the samples were also assayed using SDS-SEC, on both reducedand non-reduced samples, as previously described to determine the amountof covalent dimer in the samples. The SDS-SEC chromatograms for thesamples containing either 2.5% or 12% water and stored for 2 months at50° C. are illustrated in FIGS. 17A-B, respectively. As shown in FIG.17A, Apo2L/TRAIL crystals containing 2.5% moisture contain a relativelylarge amount of covalently linked dimer after storage at 50° C. for 2months when assayed by SDS-SEC on both reduced and non-reduced samples.This finding suggests the formation of a non-thiol covalent bond as theprimary degradation at low moisture contents. While the samplecontaining 12.5% water also contains a relatively large amount of dimerby non-reduced SDS-SEC after storage under the same conditions, thedimers formed in the higher moisture-containing samples proved to beprimarily reducible or thiol-mediated (FIG. 17B). These results suggesta difference in degradation mechanism may occur in those samples having2.5% or 12.5% residual moisture.

The presence of higher molecular weight aggregrates in the samples wasmeasured using a SEC assay, as described above. The higher molecularweight species from the samples containing either 2.5% or 12% water andstored for 2 months at 50° C. were fraction collected and assayed byreduced and non-reduced SDS-SEC to determine if the covalently linkeddimers seen by the denatured SEC methods are a result of inter-trimercovalent bonding. As shown in FIG. 18A, the collected hexamer from thesample stored with 2.5% residual moisture contained an approximate ratioof 2 monomers per non-reducible dimer with no significant amounts ofreducible dimer. Conversely, as shown in FIG. 18B, the larger molecularweight aggregates formed in higher water content crystals contained amixture of both reducible and non-reducible dimer. Taken together, thesedata indicate that the non-reducible covalent dimer seen by SDS-SECmethods primarily in lower moisture content samples is a result ofinter-molecular covalent bonding to form the hexameric structure seen bySEC. The reducible dimer seen by reduced SDS-SEC in the higher moisturesamples is thought to be a result of intra-molecular disulfide bondingbetween two of the three cysteine residues contained within the trimer.

These results suggest that there may be a degradation mechanism presentwhen the Apo2L/TRAIL crystal composition contains either too low of awater or moisture content or alternatively, too high of a water ormoisture content. At low moisture content, the protein tended to form acovalently linked hexamer species but not intra-molecular disulfidebonds. As the moisture content was increased, the rate of formation ofthis species decreased, however, the rate of intra-molecular disulfidebonds increased. It is believed that it may be desirable to achieve abalance between hexamer formation and thiol mediated intra-moleculardimer formation, and that a range of about 5% to about 10% water in theprotein crystal composition may optimally achieve such a balance. (See,e.g., FIGS. 19A-B)

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

1. A method of making a stable formulation of Apo-2 ligand, comprisingsteps of (a) providing about 1 mg/ml to about 20 mg/ml Apo-2 ligand,about 0.2 M to about 0.5M arginine salt, buffer, and surfactant, (b)combining or mixing the ingredients of step (a) to make a formulation,and (c) adjusting the pH of the formulation of step (b) to about 6 toabout
 9. 2. The method of claim 1, wherein said arginine salt isarginine succinate.
 3. The method of claim 2, wherein the concentrationof said arginine succinate is about 0.4M to about 0.5M.
 4. The method ofclaim 1, wherein said buffer is Tris buffer.
 5. The method of claim 1,wherein said surfactant is a polysorbate.
 6. The method of claim 1,wherein said Apo-2 ligand comprises amino acids 114 to 281 of SEQ. ID.NO:
 1. 7. The method of claim 1, wherein the arginine salt is selectedfrom the group consisting of arginine succinate, arginine sulphate,arginine malate, arginine citrate, arginine tartrate, and argininephosphate.
 8. The method of claim 1, wherein said formulation islyophilized.
 9. The method of claim 1, wherein the pH of saidformulation is about 6.5 to about 8.5.
 10. The method of claim 9,wherein the pH of said formulation is about 7 to about 7.5.
 11. Themethod of claim 1, wherein said Apo-2 ligand is not linked or fused toan epitope tag.
 12. The method of claim 1, wherein step (a) furthercomprises providing a preservative.
 13. The method of claim 1, whereinsaid formulation is storage-stable for at least 12 months.
 14. Themethod of claim 13, wherein said formulation is storage-stable for atleast 24 months.